U.S. patent application number 10/072666 was filed with the patent office on 2003-08-14 for detection method using dissociated rolling circle amplification.
Invention is credited to Abarzua, Patricio, Egholm, Michael, Kumar, Gyanendra.
Application Number | 20030152932 10/072666 |
Document ID | / |
Family ID | 27659528 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030152932 |
Kind Code |
A1 |
Kumar, Gyanendra ; et
al. |
August 14, 2003 |
Detection method using dissociated rolling circle amplification
Abstract
Disclosed are compositions and methods for detecting small
quantities of analytes such as proteins and peptides. The method
involves associating a DNA circle with the analyte and subsequent
release and rolling circle replication of the circular DNA
molecule. In the method, an amplification target circle is
associated with analytes using a conjugate of the circle and a
specific binding molecule that is specific for the analyte to be
detected. Amplification target circles not associated with the
proteins are removed, the amplification target circles that are
associated with the proteins are decoupled from the specific
binding molecule and amplified by rolling circle amplification. The
amplification is isothermic and can result in the production of a
large amount of nucleic acid from each primer. The amplified DNA
serves as a readily detectable signal for the analytes.
Inventors: |
Kumar, Gyanendra; (Guilford,
CT) ; Abarzua, Patricio; (West Caldwell, NJ) ;
Egholm, Michael; (Woodbridge, CT) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
Suite 1200
The Candler Building
127 Peachtree Street, N.E.
Atlanta
GA
30303-1811
US
|
Family ID: |
27659528 |
Appl. No.: |
10/072666 |
Filed: |
February 8, 2002 |
Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12Q 2531/125 20130101;
C12Q 2563/179 20130101; G01N 33/537 20130101; C12Q 1/6804 20130101;
C12Q 1/6804 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
We claim:
1. A method for detecting one or more analytes, the method
comprising (a) bringing into contact one or more analyte samples
and one or more reporter binding molecules, wherein each reporter
binding molecule comprises a specific binding molecule and an
amplification target circle, wherein each specific binding molecule
interacts with an analyte directly or indirectly, incubating the
analyte samples and the reporter binding molecules under conditions
that promote interaction of the specific binding molecules and
analytes, and separating the specific binding molecules that
interact with the analytes from the specific binding molecules that
do not interact with the analytes, (b) decoupling the amplification
target circles from the reporter binding molecules that interact
with the analytes, (c) bringing into contact the amplification
target circles and one or more rolling circle replication primers,
wherein the amplification target circles each comprise a
single-stranded, circular DNA molecule comprising a primer
complement portion, wherein the primer complement portion is
complementary to at least one of the rolling circle replication
primers, and incubating the rolling circle replication primers and
amplification target circles under conditions that promote
hybridization between the amplification target circles and the
rolling circle replication primers, (d) incubating the rolling
circle replication primers and amplification target circles under
conditions that promote replication of the amplification target
circles, wherein replication of the amplification target circles
results in the formation of tandem sequence DNA, wherein detection
of tandem sequence DNA indicates the presence of the corresponding
analytes.
2. The method of claim 1, wherein at least one of the reporter
binding molecules further comprises a circle capture probe, wherein
the amplification target circle of the reporter binding molecule is
associated with the reporter binding molecule via a non-covalent
interaction with the circle capture probe.
3. The method of claim 2, wherein the non-covalent interaction is
base pairing.
4. The method of claim 3, wherein decoupling of the amplification
target circle is accomplished by disrupting the base pairing.
5. The method of claim 4, wherein the base pairing is disrupted by
heating the reporter binding molecules.
6. The method of claim 2, wherein the circle capture probe
comprises an oligonucleotide.
7. The method of claim 6, wherein the oligonucleotide cannot be
extended.
8. The method of claim 7, wherein the oligonucleotide comprises a
3' end and a 5' end, wherein only the 5' end is free.
9. The method of claim 8, wherein the oligonucleotide is coupled to
the specific binding molecule of the reporter binding molecule via
the 3' end of the oligonucleotide.
10. The method of claim 8, wherein the 3' end of the
oligonucleotide is blocked.
11. The method of claim 7, wherein the oligonucleotide is
blocked.
12. The method of claim 1, wherein at least one of the reporter
binding molecules further comprises a circle linker, wherein the
amplification target circle of the reporter binding molecule is
coupled to the reporter binding molecule via the circle linker.
13. The method of claim 12, wherein the circle linker comprises a
cleavable bond.
14. The method of claim 13, wherein decoupling of the amplification
target circle is accomplished by cleaving the cleavable bond.
15. The method of claim 14, wherein the cleavable bond is cleaved
by treatment with a reducing agent.
16. The method of claim 15, wherein the cleavable bond is a
disulfide bond.
17. The method of claim 16, wherein the circle linker comprises
dithiobis succinimidyl propionate, dimethyl
3,3'-dithiobispropionimidate, dithio-bis-maleimidoethane,
3,3'-dithiobis sulfosuccinimidyl propionate, succinimidyl
6-[3-(2-pyridyldithio)-propionamido]hexonate, or N-succinimidyl
3-[2-pyridyldithio]propionate.
18. The method of claim 14, wherein the cleavable bond is cleaved
by treatment with periodate.
19. The method of claim 18, wherein the cleavable bond is a
dihydroxy bond.
20. The method of claim 19, wherein the circle linker comprises 1,4
bis-maleimidyl-2,3-dihydroxybutane, disuccinimidyl tartrate, or
disulfosuccinimidyl tartrate.
21. The method of claim 12, wherein the circle linker is coupled to
the amplification target circle via a reactive group on the
amplification target circle.
22. The method of claim 21, wherein the reactive group is an allyl
amino group.
23. The method of claim 1, wherein a plurality of reporter binding
molecules are brought into contact with the one or more analyte
samples.
24. The method of claim 1, wherein a plurality of analyte samples
are brought into contact with the one or more reporter binding
molecules.
25. The method of claim 1, wherein at least one of the analytes is
a protein or peptide.
26. The method of claim 1, wherein at least one of the analytes is
a lipid, glycolipid, or proteoglycan.
27. The method of claim 1, wherein at least one of the analytes is
from a human source.
28. The method of claim 1, wherein at least one of the analytes is
from a non-human source.
29. The method of claim 1, wherein none of the analytes are nucleic
acids.
30. The method of claim 1, wherein the specific binding molecules
that interact with the analytes are separated by bringing into
contact at least one of the analyte samples and one or more analyte
capture agents, wherein each analyte capture agent interacts with
an analyte directly or indirectly, wherein at least one analyte, if
present in the analyte sample, interacts with at least one analyte
capture agent, and separating analyte capture agents from the
analyte samples, thus separating specific binding molecules that
interact with the analytes from the analyte samples.
31. The method of claim 30, wherein at least one analyte capture
agent is associated with a solid support, wherein analytes that
interact with the analyte capture agent associated with a solid
support become associated with the solid support.
32. The method of claim 31, wherein the solid support comprise one
or more reaction chambers, wherein a plurality of the analyte
capture agents are located in the same reaction chamber on the
solid support.
33. The method of claim 31, wherein a plurality of reporter binding
molecules are brought into contact with one or more analyte
samples, wherein two or more of the amplification target circles
are replicated in the same reaction chamber of the solid support,
wherein the amplification target circles replicated in the same
reaction chamber of the solid support are different, wherein each
different amplification target circle produces a different tandem
sequence DNA, wherein the presence or absence of different analytes
is indicated by the presence or absence of corresponding tandem
sequence DNA.
34. The method of claim 33, wherein replication of each different
amplification target circle is primed by a different one of the
rolling circle replication primers.
35. The method of claim 31, wherein the solid support comprises
acrylamide, agarose, cellulose, cellulose, nitrocellulose, glass,
gold, polystyrene, polyethylene vinyl acetate, polypropylene,
polymethacrylate, polyethylene, polyethylene oxide, glass,
polysilicates, polycarbonates, teflon, fluorocarbons, nylon,
silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, functionalized silane, polypropylfumerate,
collagen, glycosaminoglycans, or polyamino acids.
36. The method of claim 30, further comprising bringing into
contact at least one of the analyte samples and at least one of the
reporter binding molecules with at least one accessory molecule,
wherein the accessory molecule affects the interaction of at least
one of the analytes and at least one of the specific binding
molecules or at least one of the analyte capture agents.
37. The method of claim 36, wherein the accessory molecule is
brought into contact with at least one of the analyte samples, at
least one of the reporter binding molecules, or both, prior to,
simultaneous with, or following step (a).
38. The method of claim 36, wherein at least one analyte capture
agent is associated with a solid support, wherein the accessory
molecule is associated with the solid support.
39. The method of claim 38, wherein the accessory molecule is
associated with the solid support by bringing the accessory
molecule into contact with the solid support prior to, simultaneous
with, or following step (a).
40. The method of claim 36, wherein the accessory molecule is a
protein kinase, a protein phosphatase, an enzyme, or a
compound.
41. The method of claim 36, wherein the accessory molecule is a
molecule of interest, wherein one or more of the analytes are test
molecules, wherein interactions of the test molecules with the
molecule of interest are detected.
42. The method of claim 36, wherein at least one of the analytes is
a molecule of interest, wherein the accessory molecule is a test
molecule, wherein interactions of the test molecule with the
molecule of interest are detected.
43. The method of claim 30, wherein the analyte samples include one
or more first analyte samples and one or more second analyte
samples, wherein the reporter binding molecules include one or more
first reporter binding molecules and one or more second reporter
binding molecules, the method further comprising, following step
(a) and prior to bringing the analyte samples and the solid support
into contact, mixing one or more of the first analyte samples and
one or more of the second analyte samples, wherein for each first
reporter binding molecule there is a matching second reporter
binding molecule, wherein the specific binding molecules of the
first reporter binding molecules interacts with the same analyte as
the specific binding molecules of the matching second reporter
binding molecule, wherein the amplification target circle of each
different reporter binding molecule is different, wherein each
different amplification target circle produces a different tandem
sequence DNA, wherein the presence or absence of the same analyte
in different analyte samples is indicated by the presence or
absence of corresponding tandem sequence DNA.
44. The method of claim 43, wherein replication of each different
amplification target circle is primed by a different one of the
rolling circle replication primers.
45. The method of claim 44, wherein the tandem sequence DNA
corresponding to one of the analytes and produced in association
with a first reporter binding molecule is in the same location on
the solid support as tandem sequence DNA corresponding to the same
analyte and produced in association with the matching second
reporter binding molecule, wherein the presence or absence of the
same analyte in different analyte samples is indicated by the
presence or absence of corresponding tandem sequence DNA.
46. The method of claim 30, wherein at least one of the analyte
capture agents is a molecule of interest, wherein one or more of
the analytes are test molecules, wherein interactions of the test
molecules with the molecule of interest are detected.
47. The method of claim 30, wherein at least one of the analytes is
a molecule of interest, wherein one or more of the analyte capture
agents are test molecules, wherein interactions of the test
molecules with the molecule of interest are detected.
48. The method of claim 1, wherein a plurality of reporter binding
molecules are brought into contact with one or more analyte
samples, wherein two or more of the amplification target circles
are replicated in the same reaction, wherein the amplification
target circles replicated in the same reaction are different,
wherein each different amplification target circle produces a
different tandem sequence DNA, wherein the presence or absence of
different analytes is indicated by the presence or absence of
corresponding tandem sequence DNA.
49. The method of claim 48, wherein replication of each different
amplification target circle is primed by a different one of the
rolling circle replication primers.
50. The method of claim 1, further comprising, prior to,
simultaneous with, or following step (a), bringing into contact one
or more first analyte capture agents and one or more first analyte
samples, and bringing into contact one or more second analyte
capture agents and one or more second analyte samples, wherein each
analyte capture agent comprises an analyte interaction portion and
a capture portion, wherein for each first analyte capture agent
there is a matching second analyte capture agent, wherein the
analyte interaction portions of the first analyte capture agents
interact with the same analyte as the analyte interaction portions
of the matching second analyte capture agents, wherein the capture
portions of the first and second analyte capture agents each
interact with a specific binding molecule of one or more of the
reporter binding molecules, wherein the capture portions of the
first analyte capture agents interact with different specific
binding molecules than the capture portions of the matching second
analyte capture agents, wherein each different specific binding
molecule is part of a different one of the reporter binding
molecules, wherein the amplification target circle of each
different reporter binding molecule is different, wherein
replication of each different amplification target circle is primed
by a different one of the rolling circle replication primers,
wherein each different amplification target circle produces a
different tandem sequence DNA, wherein the amplification target
circle of a reporter binding molecule that comprises a specific
binding molecule that interacts with an analyte capture agent
corresponds to the analyte capture agent, wherein the presence or
absence of the same analyte in different analyte samples is
indicated by the presence or absence of corresponding tandem
sequence DNA.
51. The method of claim 50, further comprising mixing one or more
of the first analyte samples and one or more of the second analyte
samples.
52. The method of claim 50, further comprising mixing the one or
more first analyte capture agents and the one or more second
analyte capture agents.
53. The method of claim 52, wherein mixing the one or more first
analyte capture agents and the one or more second analyte capture
agents is accomplished by associating, simultaneously or
sequentially, the one or more first analyte capture agents and the
one or more second analyte capture agents with the same solid
support.
54. The method of claim 50, wherein the tandem sequence DNA
corresponding to one of the analytes and produced in association
with a first analyte capture agent is in the same location as, and
is simultaneously detected with, tandem sequence DNA corresponding
to the same analyte and produced in association with the matching
second analyte capture agent, wherein the presence or absence of
the same analyte in different analyte samples is indicated by the
presence or absence of corresponding tandem sequence DNA.
55. The method of claim 50, wherein the capture portion of each
first analyte capture agent is the same, wherein the reporter
binding molecules corresponding to the first analyte capture agents
are the same, wherein the amplification target circles
corresponding to the first analyte capture agents are the same,
wherein the capture portion of each second analyte capture agent is
the same, wherein the reporter binding molecules corresponding to
the second analyte capture agents are the same, wherein the
amplification target circles corresponding to the second analyte
capture agents are the same.
56. The method of claim 1, wherein at least one of the specific
binding molecules is an antibody specific for at least one of the
analytes.
57. The method of claim 1, wherein at least one of the specific
binding molecules is a molecule that specifically binds to at least
one of the analytes.
58. The method of claim 1, wherein at least one of the specific
binding molecules is a molecule that specifically binds to at least
one of the analytes in combination with an accessory molecule.
59. The method of claim 1, wherein the specific binding molecules
and analytes interact by binding to each other directly or
indirectly.
60. The method of claim 1, wherein at least one accessory molecule
is brought into contact with at least one of the analyte samples
and at least one of the reporter binding molecules, wherein the
accessory molecule affects the interaction of at least one of the
analytes and at least one of the specific binding molecules or at
least one of the analyte capture agents.
61. The method of claim 60, wherein the accessory molecule competes
with the interaction of at least one of the specific binding
molecules or at least one of the analyte capture agents.
62. The method of claim 61, wherein the accessory molecule is an
analog of at least one of the analytes.
63. The method of claim 60, wherein the accessory molecule
facilitates the interaction of at least one of the specific binding
molecules or at least one of the analyte capture agents.
64. The method of claim 60, wherein the accessory molecule is
brought into contact with at least one of the analyte samples, at
least one of the reporter binding molecules, or both, prior to,
simultaneous with, or following step (a).
65. The method of claim 60, wherein the accessory molecule is a
protein kinase, a protein phosphatase, an enzyme, or a
compound.
66. The method of claim 60, wherein the accessory molecule is at
least 20% pure.
67. The method of claim 60, wherein the accessory molecule is at
least 50% pure.
68. The method of claim 60, wherein the accessory molecule is at
least 80% pure.
69. The method of claim 60, wherein the accessory molecule is at
least 90% pure.
70. The method of claim 1, wherein at least one of the analytes is
associated with a solid support.
71. The method of claim 70, wherein the solid support comprises one
or more reaction chambers, wherein a plurality of the analytes
associated with the solid support are associated with the solid
support in the same reaction chamber.
72. The method of claim 70, wherein at least one of the analytes
associated with the solid support is associated with the solid
support indirectly.
73. The method of claim 72, wherein the analytes associated with
the solid support interact with analyte capture agents, and wherein
the analyte capture agents are associated with the solid support
thereby indirectly associating the analytes with the solid
support.
74. The method of claim 1, wherein at least one specific binding
molecule interacts with at least one analyte indirectly.
75. The method of claim 74, wherein the analyte interacts with an
analyte capture agent, and wherein the specific binding molecule
interacts with the analyte capture agent thereby indirectly
associating the specific binding molecule with the analyte.
76. The method of claim 1, wherein at least one of the analytes is
a modified form of another analyte, wherein the specific binding
molecule of at least one of the reporter binding molecules
interacts, directly or indirectly, with the analyte that is a
modified form of the other analyte, and wherein the specific
binding molecule of another reporter binding molecule interacts,
directly or indirectly, with the other analyte.
77. The method of claim 76, wherein the analytes are proteins,
wherein the modification of the modified form of the other analyte
is a post-translational modification.
78. The method of claim 77, wherein the modification is
phosphorylation or glycosylation.
79. The method of claim 1, wherein detection of the tandem sequence
DNA is accomplished by mixing a set of detection probes with the
tandem sequence DNA under conditions that promote hybridization
between the tandem sequence DNA and the detection probes.
80. The method of claim 79, wherein a plurality of different tandem
sequence DNAs are detected separately and simultaneously via
multiplex detection.
81. The method of claim 80, wherein the set of detection probes is
labeled using combinatorial multicolor coding.
82. The method of claim 1, further comprising, simultaneous with,
or following, step (d), bringing into contact a secondary DNA
strand displacement primer and the tandem sequence DNA, and
incubating under conditions that promote (i) hybridization between
the tandem sequence DNA and the secondary DNA strand displacement
primer, and (ii) replication of the tandem sequence DNA, wherein
replication of the tandem sequence DNA results in the formation of
secondary tandem sequence DNA.
83. The method of claim 82, wherein the tandem sequence DNA,
secondary tandem sequence DNA, or both, are detected during
replication of the amplification target circles.
84. The method of claim 83, wherein the tandem sequence DNA,
secondary tandem sequence DNA, or both, are detected by detecting
fluorescent moieties incorporated into the tandem sequence DNA,
secondary tandem sequence DNA, or both.
85. The method of claim 82, wherein the tandem sequence DNA,
secondary tandem sequence DNA, or both, are detected during
replication of the tandem sequence DNA.
86. The method of claim 82, wherein at least one of the rolling
circle replication primers is a fluorescent quenched primer.
87. The method of claim 82, wherein at least one of the secondary
DNA strand displacement primers is a fluorescent quenched
primer.
88. The method of claim 82, wherein at least one of the rolling
circle replication primers and at least one of the secondary DNA
strand displacement primers are fluorescent quenched primers.
89. The method of claim 82, wherein the tandem sequence DNA,
secondary tandem sequence DNA, or both, are detected by detecting
fluorescent moieties incorporated into the tandem sequence DNA.
90. The method of claim 82, wherein the secondary tandem sequence
DNA is replicated to form higher order tandem sequence DNA.
91. The method of claim 90, wherein the amplification target
circles, the tandem sequence DNA, and the secondary tandem sequence
DNA are replicated simultaneously.
92. The method of claim 90, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected during replication of the amplification
target circles.
93. The method of claim 90, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected by detecting fluorescent moieties
incorporated into the tandem sequence DNA, secondary tandem
sequence DNA, higher order tandem sequence DNA, or a
combination.
94. The method of claim 90, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected during replication of the tandem
sequence DNA.
95. The method of claim 90, wherein at least one of the rolling
circle replication primers is a fluorescent quenched primer.
96. The method of claim 90, wherein at least one of the secondary
DNA strand displacement primers is a fluorescent quenched
primer.
97. The method of claim 90, wherein at least one of the rolling
circle replication primers and at least one of the secondary DNA
strand displacement primers are fluorescent quenched primers.
98. The method of claim 90, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected by detecting fluorescent moieties
incorporated into the tandem sequence DNA.
99. The method of claim 1, wherein the tandem sequence DNA is
detected during replication of the amplification target
circles.
100. The method of claim 99, wherein the tandem sequence DNA is
detected by detecting fluorescent moieties incorporated into the
tandem sequence DNA.
101. The method of claim 1, wherein at least one of the rolling
circle replication primers is a fluorescent quenched primer.
102. The method of claim 1, wherein the tandem sequence DNA is
detected by detecting fluorescent moieties incorporated into the
tandem sequence DNA.
103. The method of claim 1, wherein the reporter binding molecules
are at least 20% pure.
104. The method of claim 1, wherein the reporter binding molecules
are at least 50% pure.
105. The method of claim 1, wherein the reporter binding molecules
are at least 80% pure.
106. The method of claim 1, wherein the reporter binding molecules
are at least 90% pure.
107. A method for detecting one or more analytes, the method
comprising (a) bringing into contact one or more analyte samples
and one or more analyte capture agents, wherein each analyte
capture agent interacts with an analyte directly or indirectly,
wherein at least one analyte, if present in the analyte sample,
interacts with at least one analyte capture agent, incubating the
analyte samples and the analyte capture agents under conditions
that promote interaction of the analyte capture agents and
analytes, (b) bringing into contact at least one of the analyte
samples and one or more reporter binding molecules, wherein each
reporter binding molecule comprises a specific binding molecule and
an amplification target circle, wherein each specific binding
molecule interacts with an analyte capture agent directly or
indirectly, incubating the analyte samples and the reporter binding
molecules under conditions that promote interaction of the specific
binding molecules and analyte capture agents, and separating the
specific binding molecules that interact with the analyte capture
agents from the specific binding molecules that do not interact
with the analyte capture agents, (c) decoupling the amplification
target circles from the reporter binding molecules that interact
with the analyte capture agents, (d) bringing into contact the
amplification target circles and one or more rolling circle
replication primers, wherein the amplification target circles each
comprise a single-stranded, circular DNA molecule comprising a
primer complement portion, wherein the primer complement portion is
complementary to at least one of the rolling circle replication
primers, and incubating the rolling circle replication primers and
amplification target circles under conditions that promote
hybridization between the amplification target circles and the
rolling circle replication primers, (e) incubating the rolling
circle replication primers and amplification target circles under
conditions that promote replication of the amplification target
circles, wherein replication of the amplification target circles
results in the formation of tandem sequence DNA, wherein detection
of tandem sequence DNA indicates the presence of the corresponding
analytes.
108. A method for detecting one or more analytes, the method
comprising (a) treating one or more analyte samples so that one or
more analytes are modified, (b) bringing into contact at least one
of the analyte samples and one or more reporter binding molecules,
wherein each reporter binding molecule comprises a specific binding
molecule and an amplification target circle, wherein each specific
binding molecule interacts with a modified analyte directly or
indirectly, incubating the analyte samples and the reporter binding
molecules under conditions that promote interaction of the specific
binding molecules and modified analytes, and separating the
specific binding molecules that interact with the modified analytes
from the specific binding molecules that do not interact with the
modified analytes, (c) decoupling the amplification target circles
from the reporter binding molecules that interact with the modified
analytes, (d) bringing into contact the amplification target
circles and one or more rolling circle replication primers, wherein
the amplification target circles each comprise a single-stranded,
circular DNA molecule comprising a primer complement portion,
wherein the primer complement portion is complementary to at least
one of the rolling circle replication primers, and incubating the
rolling circle replication primers and amplification target circles
under conditions that promote hybridization between the
amplification target circles and the rolling circle replication
primers, (e) incubating the rolling circle replication primers and
amplification target circles under conditions that promote
replication of the amplification target circles, wherein
replication of the amplification target circles results in the
formation of tandem sequence DNA, wherein detection of tandem
sequence DNA indicates the presence of the corresponding modified
analytes.
109. The method of claim 108, wherein all of the analytes are
modified by associating a modifying group to the analytes, wherein
the modifying group is the same for all of the analytes, wherein
all of the specific binding molecules interact with the modifying
group.
110. A method for detecting one or more analytes, the method
comprising (a) bringing into contact one or more analyte samples
and a set of analyte capture agents, a set of accessory molecules,
or both, wherein each analyte capture agent interacts with an
analyte directly or indirectly, (b) prior to, simultaneous with, or
following step (a), bringing into contact at least one of the
analyte samples and one or more reporter binding molecules, wherein
each reporter binding molecule comprises a specific binding
molecule and an amplification target circle, wherein each specific
binding molecule interacts with an analyte directly or indirectly,
wherein each accessory molecule affects the interaction of at least
one of the analytes and at least one of the specific binding
molecules or at least one of the analyte capture agents, (c)
simultaneous with, or following, steps (a) and (b), incubating the
analyte samples, the analyte capture agents, the accessory
molecules, and the reporter binding molecules under conditions that
promote interaction of the specific binding molecules, analytes,
analyte capture agents, and accessory molecules, and separating the
specific binding molecules that interact with the analytes from the
specific binding molecules that do no t interact with the analytes,
decoupling the amplification target circles from the reporter
binding molecules that interact with the analytes, (d) bringing
into contact the amplification target circles and one or more
rolling circle replication primers, wherein the amplification
target circles each comprise a single-stranded, circular DNA
molecule comprising a primer complement portion, wherein the primer
complement portion is complementary to at least one of the rolling
circle replication primers, and incubating the rolling circle
replication primers and amplification target circles under
conditions that promote hybridization between the amplification
target circles and the rolling circle replication primers, (e)
incubating the reporter binding molecules and amplification target
circles under conditions that promote replication of the
amplification target circles, wherein replication of the
amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
111. The method of claim 110, wherein the analyte capture agents
are immobilized on a solid support, wherein the solid support
comprises one or more reaction chambers, wherein a plurality of the
analyte capture agents are immobilized in the same reaction chamber
of the solid support.
112. The method of claim 111, wherein the analyte capture agents
are immobilized to the solid support at a density exceeding 400
different analyte capture agents per cubic centimeter.
113. The method of claim 111, wherein the analyte capture agents
are peptides.
114. The method of claim 113, wherein each of the different
peptides is at least 4 amino acids in length.
115. The method of claim 114, wherein each different peptide is
from about 4 to about 20 amino acids in length.
116. The method of claim 114, wherein each different peptide is at
least 10 amino acids in length.
117. The method of claim 114, wherein each different peptide is at
least 20 amino acids in length.
118. The method of claim 111, wherein the solid support comprises a
plurality of reaction chambers.
119. The method of claim 111, wherein the solid support comprises
acrylamide, agarose, cellulose, cellulose, nitrocellulose, glass,
gold, polystyrene, polyethylene vinyl acetate, polypropylene,
polymethacrylate, polyethylene, polyethylene oxide, glass,
polysilicates, polycarbonates, teflon, fluorocarbons, nylon,
silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, functionalized silane, polypropylfumerate,
collagen, glycosaminoglycans, or polyamino acids.
120. The method of claim 111 , wherein the analyte capture agents
in the reaction chambers are at least 20% pure.
121. The method of claim 111, wherein the analyte capture agents in
the reaction chambers are at least 50% pure.
122. The method of claim 111, wherein the analyte capture agents in
the reaction chambers are at least 80% pure.
123. The method of claim 111, wherein the analyte capture agents in
the reaction chambers are at least 90% pure.
124. A method for detecting one or more analytes, the method
comprising bringing into contact one or more analyte samples and
one or more reporter binding molecules, wherein each reporter
binding molecule comprises a specific binding molecule and an
amplification target circle, wherein each specific binding molecule
can interact with an analyte directly or indirectly, separating the
specific binding molecules that interact with the analytes from the
specific binding molecules that do not interact with the analytes,
decoupling the amplification target circles from the reporter
binding molecules that interact with the analytes, replicating the
amplification target circles, wherein replication of the
amplification target circles results in the formation of tandem
sequence DNA, secondary tandem sequence DNA, and higher order
tandem sequence DNA, wherein detection of tandem sequence DNA,
secondary tandem sequence DNA, and higher order tandem sequence
DNA, or a combination, indicates the presence of the corresponding
analytes.
125. The method of claim 124, wherein the amplification target
circles, the tandem sequence DNA, and the secondary tandem sequence
DNA are replicated simultaneously.
126. The method of claim 124, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected during replication of the amplification
target circles.
127. The method of claim 124, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected by detecting fluorescent moieties
incorporated into the tandem sequence DNA, secondary tandem
sequence DNA, higher order tandem sequence DNA, or a
combination.
128. The method of claim 124, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected during replication of the tandem
sequence DNA.
129. The method of claim 124, wherein at least one of the rolling
circle replication primers is a fluorescent quenched primer.
130. The method of claim 124, wherein at least one of the secondary
DNA strand displacement primers is a fluorescent quenched
primer.
131. The method of claim 124, wherein at least one of the rolling
circle replication primers and at least one of the secondary DNA
strand displacement primers are fluorescent quenched primers.
132. The method of claim 124, wherein the tandem sequence DNA,
secondary tandem sequence DNA, higher order tandem sequence DNA, or
a combination, is detected by detecting fluorescent moieties
incorporated into the tandem sequence DNA.
133. A method for detecting one or more analytes, the method
comprising bringing into contact one or more analyte samples and
one or more reporter binding molecules, wherein each reporter
binding molecule comprises a specific binding molecule and an
amplification target circle, wherein each specific binding molecule
can interact with an analyte directly or indirectly, separating the
specific binding molecules that interact with the analytes from the
specific binding molecules that do no t interact with the an
analytes, decoupling the amplification target circles from the
reporter binding molecules that interact with the analytes,
replicating the amplification target circles, wherein replication
of the amplification target circles results in the formation of
tandem sequence DNA, wherein detection of tandem sequence DNA
indicates the presence of the corresponding analytes.
134. A method for detecting one or more analytes, the method
comprising bringing into contact one or more analyte samples and
one or more analyte capture agents, wherein each analyte capture
agents can interact with an analyte directly or indirectly,
bringing into contact at least one of the analyte samples and one
or more reporter binding molecules, wherein each reporter binding
molecule comprises a specific binding molecule and an amplification
target circle, wherein each specific binding molecule can interact
with an analyte capture agent directly or indirectly, separating
the specific binding molecules that interact with the analyte
capture agents from the specific binding molecules that do not
interact with the analyte capture agents, decoupling the
amplification target circles from the reporter binding molecules
that interact with the analyte capture agents, replicating the
amplification target circles, wherein replication of the
amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
135. A method for detecting one or more analytes, the method
comprising treating one or more analyte samples so that one or more
analytes are modified, bringing into contact at least one analyte
samples and one or more reporter binding molecules, wherein each
reporter binding molecule comprises a specific binding molecule and
an amplification target circle, wherein each specific binding
molecule can interact with a modified analyte directly or
indirectly, separating the specific binding molecules that interact
with the modified analytes from the specific binding molecules that
do not interact with the modified analytes, decoupling the
amplification target circles from the reporter binding molecules
that interact with the modified analytes, replicating the
amplification target circles, wherein replication of the
amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding modified analytes.
136. A method for detecting one or more analytes, the method
comprising bringing into contact one or more analyte samples and a
set of analyte capture agents, a set of accessory molecules, or
both, wherein each analyte capture agent can interact with an
analyte directly or indirectly, bringing into contact at least one
of the analyte samples and one or more reporter binding molecules,
wherein each reporter binding molecule comprises a specific binding
molecule and an amplification target circle, wherein each specific
binding molecule can interact with an analyte directly or
indirectly, wherein each accessory molecule can affect the
interaction of at least one of the analytes and at least one of the
specific binding molecules or at least one of the analyte capture
agents, separating the specific binding molecules that interact
with the analytes from the specific binding molecules that do not
interact with the analytes, decoupling the amplification target
circles from the reporter binding molecules that interact with the
analytes, replicating the amplification target circles, wherein
replication of the amplification target circles results in the
formation of tandem sequence DNA, wherein detection of tandem
sequence DNA indicates the presence of the corresponding
analytes.
137. A kit comprising (a) a plurality of reporter binding
molecules, wherein each reporter binding molecule comprises a
specific binding molecule and an amplification target circle,
wherein the amplification target circle can be decoupled from the
reporter binding molecule, wherein each specific binding molecule
interacts with an analyte directly or indirectly, and (b) a
plurality of analyte capture agents, wherein each analyte capture
agent interacts with an analyte directly or indirectly.
138. The kit of claim 137, wherein the analyte capture agents are
associated with a solid support.
Description
FIELD OF THE INVENTION
[0001] The disclosed invention is generally in the area of
detection of analytes, and specifically in the area of detection of
analytes using rolling circle amplification.
BACKGROUND OF THE INVENTION
[0002] The information content of the genome is carried as
deoxyribonucleic acid (DNA). The size and composition of a given
genomic sequence determines the form and function of the resultant
organism. In general, genomic complexity is proportional to the
complexity of the organism. Relatively simple organisms such as
bacteria have genomes of about 1-5 million megabases while
mammalian genomes are approximately 3000 megabases. The genome is
generally divided into distinct segments known as chromosomes. The
bacterium Escherichia coli (E. coli) contains a single circular
chromosome, whereas the human genome consists of 24
chromosomes.
[0003] Genomic DNA exists as a double-stranded polymer containing
four DNA bases (A, G, C, and T) tethered to a sugar-phosphate
backbone. The order of the bases along the DNA is the primary
sequence of the DNA. The genome of an organism contains both
protein coding and non-coding regions, including exons and introns,
promoter and gene regulatory regions, and non-functional DNA.
Genome analysis can provide a quantitative measure of gene copy
number and chromosome number, as well as the presence of single
base differences in the primary sequence of the DNA. Single base
changes that are inherited are referred to as polymorphisms,
whereas those that are acquired during the life of an organism are
known as mutations. Genomic analysis at the DNA level does not
provide a measure of gene expression (that is, the process by which
RNA and protein copies of the coding sequences are
synthesized).
[0004] All of the cells from a given organism are assumed to
contain identical genomes, while genomes from different individuals
of the same species are typically about 99.9% identical. The 0.1%
polymorphism rate among individuals (Wang et al., Science 280: 1077
(1998)) is significant in that approximately three million
polymorphisms are expected to be found upon complete sequencing of
any two human genomes. If single base changes occur in protein
coding segments, polymorphisms can alter the protein sequence and
therefore change the biochemical activity of the protein.
[0005] The DNA genome consists of discrete functional regions known
as genes. Genomes of simple organisms such as bacteria contain
approximately 1000 genes (Fleischmann et al., Science 269: 496
(1995)), whereas the human genome is estimated to contain about
100,000 genes (Fields et al., Nature Genet. 7: 345 (1994)). Genomic
analysis at the mRNA level can be used as a measure of gene
expression. Expression levels for each gene are determined by a
combination of genetic and environmental factors. The genetic
factors include the precise DNA sequence of gene regulatory regions
such as promoters, enhancers, and splice sites. Polymorphisms in
the DNA are thus expected to contribute some of the differences in
gene expression among individuals of the same species. Expression
levels are also affected by environmental factors, including
temperature, stress, light, and signals that lead to changes in the
levels of hormones and other signaling substances. For this reason,
RNA analysis provides information not only about the genetic
potential of an organism, but also about changes in functional
state (M. Schena and R. W. Davis, DNA Microarrays: A Practical
Approach. (Oxford University Press, New York, 1999) 1-16.)
[0006] The second step in gene expression is the synthesis of
protein from mRNA. A unique protein is encoded by each mRNA, such
that every three nucleotides of mRNA encodes one amino acid of the
polypeptide chain, with the linear order of the nucleotides
represented as a linear sequence of amino acids. Once synthesized,
the protein assumes a unique three-dimensional conformation that is
determined largely by the primary amino acid sequence. Proteins
impart the functional instructions of the genome by performing a
wide range of biochemical activities including roles in gene
regulation, metabolism, cell structure, and DNA replication.
[0007] Individuals in a population may have differences in protein
activity due to polymorphisms that either alter the primary amino
acid sequence of the proteins or perturb steady state protein
levels by altering gene expression. Similar to mRNA levels, protein
levels can also change in response to changes in the environment;
moreover, protein levels are also subject to translational and
post-translational control which do not effect mRNA levels directly
(Schena and David, 1999). Proteomics analysis provides data on when
or if a predicted gene product is actually translated, the level
and type of post-translational modification it may undergo and its
relative concentration compared with other proteins (Humphrey-Smith
and Blackstock, J. Protein. Chem. 16: 537-544 (1997)). After DNA is
transcribed into mRNA, the exons may be spliced in different ways
before being translated into proteins. Following the translation of
mRNA by ribosomes, proteins are usually post-translationally
modified by the addition of different chemical groups such as
carbohydrate, lipid and phosphate groups, as well as through the
proteolytic cleavage of specific peptide bonds. These chemical
modifications are crucial to modulating protein function but are
not directly coded for by genes. Furthermore, both mRNA and protein
are continually being synthesized and degraded, and thus final
levels of protein are not easily obtainable by measuring mRNA
levels (Patton, J. Chromatogr. 722: 203-223, (1999); Patton et al.,
J. Biol. Chem. 270: 21404-21410 (1995)). So while mRNA levels are
often extrapolated to indicate the levels of expressed proteins, it
is not surprising that there is little correlation between the
abundance of mRNA species and the actual amounts of proteins that
they code for (Anderson and Seilhamer, Electrophoresis 18: 533-537;
Gygi et al., Mol. Cell. Biol. 19: 1720-1730 (1999)).
[0008] A growing body of evidence suggests that changes in gene and
protein expression may correlate with the onset of a given human
disease (Schena and Davis, 1999). Proteomic analysis of disease
tissues should allow the identification of proteins whose
expression is altered in a given illness. Many small molecules may
also alter protein expression at a global level. Combining
information about altered expression in a disease state with the
changes that result from treatment with a small molecule would
provide valuable information about classes of molecules that may be
effective in combating a given disease. Proteomics thus has a role
in processes such as lead compound screening and optimization,
toxicity, pharmacodynamics, and drug efficacy.
[0009] A pivotal component of proteomics is its ability to
accurately quantify vast numbers of proteins accurately and
reproducibly. Typically, proteomics entails the simultaneous
separation of proteins from a biological sample, and the
quantitation of the relative abundance of the proteins resolved
during the separation. Proteomics currently relies heavily on
two-dimensional (2-D) gel electrophoresis. However, obtaining
information concerning global protein expression using 2-D gels is
technically difficult, and semiautomated procedures to carry out
this process are in their infancy (Patton, Biotechniques 28:
944-957 (2000)). Furthermore, the commonly used stains for
evaluating protein expression in 2-D gels (such as Coomassie Blue,
colloidal gold and silver stain) do not provide the requisite
dynamic range to be effective in this capacity. These stains are
linear over only a 10- to 40-fold range, whereas the abundance of
individual proteins differs by as much as four orders of magnitude
(Brush, The Scientist 12:16-22, 1998; Wirth and Romano, J.
Chromatogr 698: 123-143 (1995)). In addition, low abundance
proteins, such as transcription factors and kinases that are
present in 1-2000 copies per cell, often represent species that
perform important regulatory functions. The accurate detection of
such low-abundance proteins is an important challenge to
proteomics. Methods have recently been introduced to directly
quantify the relative abundance of proteins in two different
samples by mass spectrometry. However, the linear dynamic range of
these methods has been demonstrated over only a four- to ten-fold
range (Gygi et al. 1999; Oda et al., Proc. Natl. Acad. Sci USA 96:
6591-6596 (1999)).
[0010] It has been noted that developing microarray technologies
would make possible the simultaneous, ultra-sensitive measurement
of hundreds or even thousands of substances in a small sample
(Ekins, Clin. Chem. 44: 2015-2030 (1998)). This approach has been
difficult to put into practice, however, because the extremely
small volumes (about 0.5-5 nl) of sample used to create spots on
these microarrays makes it necessary to utilize methods of analyte
detection that are extremely sensitive. Rolling Circle
Amplification (RCA) driven by DNA polymerase can replicate circular
oligonucleotide probes with either linear or geometric kinetics
under isothermal conditions (Lizardi et al., Nature Genet. 19:
225-232 (1998)). If a single primer is used, RCA generates in a few
minutes a linear chain of hundreds or thousands of tandemly-linked
DNA copies of a target which is covalently linked to that target.
Generation of a linear amplification product permits both spatial
resolution and accurate quantitation of a target. DNA generated by
RCA can be labeled with fluorescent oligonucleotide tags that
hybridize at multiple sites in the tandem DNA sequences. RCA can be
used with fluorophore combinations designed for multiparametric
color coding (Speicher et al., Nature Genet. 12:368-375 (1996)),
thereby markedly increasing the number of targets that can be
analyzed simultaneously. RCA technologies can be used in solution,
in situ and in microarrays. In solid phase formats, detection and
quantitation can be achieved at the level of single molecules
(Lizardi et al., 1998).
BRIEF SUMMARY OF THE INVENTION
[0011] Disclosed are compositions and methods for detecting small
quantities of analytes such as proteins and peptides. The method
involves associating a DNA circle with the analyte and subsequent
release and rolling circle replication of the circular DNA
molecule. Thus, the disclosed method produces an amplified signal,
via rolling circle amplification, from any analyte of interest. The
amplification is isothermic and can result in the production of a
large amount of nucleic acid from each primer.
[0012] The disclosed method is preferably used to detect and
analyze proteins and peptides. In some embodiments, multiple
proteins can be analyzed using solid supports, such as microtiter
dishes, with which multiple different proteins or analytes are
directly or indirectly associated (if they are present in the
sample being tested). An amplification target circle is then
associated with the various proteins using a conjugate of the
circle and a specific binding molecule, such as an antibody, that
is specific for the protein to be detected. Amplification target
circles not associated with the proteins are removed, the
amplification target circles that are associated with the proteins
are decoupled from the specific binding molecule and replicated.
Rolling circle replication primed by rolling circle replication
primers results in production of a large amount of DNA. Use of
exponential rolling circle amplification (ERCA), where the strand
replicated from the amplification target circle is replicated using
a second primer and both replicated strands generate further
replicated strands, is preferred. Amplification products can be
detected in real time using, for example, Amplifluor.TM. primers.
The amplified DNA serves as a readily detectable signal for the
proteins. Different proteins can be distinguished in several ways.
For example, each different protein can be associated with a
different amplification target circle which in turn is replicated
to produce amplified DNA. The result is distinctive amplified DNA
for each different protein. The different amplified DNAs can be
distinguished using any suitable sequence-based nucleic acid
detection technique. In this form of the method, many proteins can
be detected in the same amplification reaction. Alternatively, the
location of the amplified DNA on a solid support can indicate the
protein involved if different proteins are immobilized at
predetermined locations on the support.
[0013] Another embodiment of the disclosed method involves
comparison of the proteins expressed in two or more different
samples. The information generated is analogous to the type of
information gathered in nucleic acid expression profiles. The
disclosed method allows sensitive and accurate detection and
quantitation of proteins expressed in any cell or tissue. The
disclosed method also allows the same analyte(s) from different
samples to be detected simultaneously in the same assay.
[0014] It is an object of the present invention to provide a method
for detecting small quantities and concentrations of analytes.
[0015] It is a further object of the present invention to provide a
method for detecting small quantities and concentrations of
multiple analytes in samples.
[0016] It is a further object of the present invention to provide a
method for amplifying the signal of an analyte to be detected.
[0017] It is a further object of the present invention to provide
an automated method for detecting small quantities and
concentrations of multiple analytes in samples.
[0018] It is a further object of the present invention to provide a
method for profiling the presence of multiple analytes in a
sample.
[0019] It is a further object of the present invention to provide a
method for comparing profiles of the presence of multiple analytes
in different samples.
[0020] It is a further object of the present invention to provide a
method for assessing the interaction of compounds with molecules of
interest.
[0021] It is a further object of the present invention to provide a
method for detecting small quantities and concentrations of
proteins and peptides.
[0022] It is a further object of the present invention to provide a
method for detecting small quantities and concentrations of
multiple proteins and peptides in samples.
[0023] It is a further object of the present invention to provide a
method for amplifying the signal of a protein or peptide to be
detected.
[0024] It is a further object of the present invention to provide
an automated method for detecting small quantities and
concentrations of multiple proteins and peptides in samples.
[0025] It is a further object of the present invention to provide a
method for profiling the presence of multiple proteins and peptides
in a sample.
[0026] It is a further object of the present invention to provide a
method for comparing profiles of the presence of multiple proteins
and peptides in different samples.
[0027] It is a further object of the present invention to provide a
method for assessing the interaction of compounds with proteins and
peptides of interest.
[0028] It is a further object of the present invention to provide
compositions for detecting small quantities and concentrations of
analytes.
[0029] It is a further object of the present invention to provide
compositions for detecting small quantities and concentrations of
proteins and peptides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIGS. 1A and 1B are diagrams of examples of two forms of the
disclosed method. In FIG. 1A, a reporter binding molecule
(anti-human IgG with circle) is associated with a protein (HIV P24
protein) via an anti-HIV P24 antibody. The protein that is attached
to Micro Amp tubes. The specific binding molecule of the reporter
binding molecule is an anti-human IgG. In FIG. 1B, a reporter
binding molecule (anti-biotin antibody with circle) is associated
with a protein (HIV P24 protein) that is associated with an
anti-HIV P24 antibody. The anti-HIV P24 antibodies are attached to
Micro Amp tubes, thus associating the protein with the Micro Amp
tubes. The specific binding molecule of the reporter binding
molecule is an anti-biotin antibody. The amplification target
circle of the reporter binding molecule is associated with the
specific binding molecule via a circle capture probe.
[0031] FIG. 2 is a graph of antibody (micrograms in 30 .mu.l)
versus absorbance at 450 nm. This shows the amount of coating by
the antibody when different amounts of antibody are used.
[0032] FIG. 3 is a diagram of a comparison of association of
reporter binding molecules to cognate and non-cognate analytes. The
"analytes" are anti-biotin antibodies (cognate) and mouse IgG
(non-cognate). The non-cognate analyte serves as a control. The
reporter binding molecules consists of biotin (the specific binding
molecule), an oligonucleotide (the circle capture probe), and an
1822 circle (the amplification target circle) which is
complementary to the oligonucleotide. The reporter binding molecule
interacts only with the anti-biotin antibodies. Decoupled
amplification target circles are amplified by ERCA using an
Amplifluor.TM. primer (P1), a secondary DNA strand displacement
primer (P2), and Bst DNA polymerase.
[0033] FIGS. 4A, 4B, and 4C are graphs of time (in "cycles," which
are 2 minute time units) versus fluorescence. The difference in
delta Ct when using different numbers of reporter binding agents is
shown between the three graphs.
[0034] FIG. 5 is a diagram of a comparison of association of
partial reporter binding molecules to cognate and non-cognate
analytes. The "analytes" are anti-biotin antibodies (cognate) and
mouse IgG (non-cognate). The non-cognate analyte serves as a
control. The partial reporter binding molecules consists of biotin
(the specific binding molecule), and an oligonucleotide (the circle
capture probe). The partial reporter binding molecule interacts
only with the anti-biotin antibodies. The amplification target
circles, which are complementary to the oligonucleotide, are
annealed to the circle capture probe after the partial reporter
binding molecule is associated with the analyte. Decoupled
amplification target circles are amplified by ERCA using an
Amplifluor.TM. primer (P1), a secondary DNA strand displacement
primer (P2), and Bst DNA polymerase.
[0035] FIG. 6 is a graph of the number of circle capture probes
used (in thousands) versus the change in counts (in minutes).
[0036] FIG. 7 is a diagram of an example of immunoRCA involving
amplification target circles associated with specific binding
molecules via base pairing to circle capture probes. Micro Amp
tubes coated with anti-IL8 antibodies (analyte capture agents) are
brought into contact with IL8 (analyte) and the IL8 binds to the
antibodies. A biotinylated anti-IL8 antibody is brought into
contact with the captured IL8 and they bind. Reporter binding
molecules (comprising an anti-biotin antibody, a circle capture
probe and an amplification target circle) are brought into contact
with the biotinylated anti-IL8 antibody and they bind. This
associates the reporter binding molecule with the analyte (IL8 )
indirectly (via the biotinylated anti-IL8 antibody). The
amplification target circle is decoupled from the reporter binding
molecule by disrupting the base pairing between the amplification
target circle and the circle capture probe and amplified in
ERCA.
[0037] FIG. 8 is a graph of the amount of IL8 (in pg/ml) versus the
change in counts (in minutes).
[0038] FIG. 9 is a diagram of an example of immunoRCA involving
amplification target circles coupled to specific binding molecules
via circle linkers having cleavable bonds. Anti-analyte antibodies
(analyte capture agents) are brought into contact with analyte and
the analyte binds to the antibodies. Biotinylated anti-analyte
antibodies are brought into contact with the captured analyte and
they bind. Reporter binding molecules (comprising an anti-biotin
antibody, a circle linker containing a cleavable bond, and an
amplification target circle) are brought into contact with the
biotinylated anti-analyte antibody and they bind. This associates
the reporter binding molecule with the analyte indirectly (via the
biotinylated anti-analyte antibody). The amplification target
circle is decoupled from the reporter binding molecule by cleaving
the cleavable bond and the circle capture probe and amplified in
ERCA.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Disclosed are compositions and methods for detecting small
quantities of analytes such as proteins and peptides. The method
applies the power of nucleic acid signal amplification to the
detection of non-nucleic acid analytes. Detection of such
analytes--for which there are no amplification techniques
comparable to nucleic acid amplification techniques--has generally
depended on detection of sufficient quantities of the analyte or
the use of extremely sensitive labels. The use of such labels is
both cumbersome and limited. The disclosed method provides a simple
and sensitive way to produce an amplified signal for any desired
analyte.
[0040] The disclosed method is a form of rolling circle
amplification (RCA) where a reporter binding molecule provides the
amplification target circle for amplification. The disclosed method
allows RCA to produce an amplified signal (that is, tandem sequence
DNA (TS-DNA)) based on association of the reporter binding molecule
with a target molecule (also referred to as an analyte). The
specific amplification target circle that is a part of the reporter
binding molecule provides the link between the specific interaction
of the reporter binding molecule to an analyte (via the affinity
portion of the reporter binding molecule) and RCA. Once the
reporter binding molecule is associated with an analyte, a rolling
circle replication primer is hybridized to the amplification target
circle (ATC) of the reporter binding molecule, followed by
amplification of the ATC by RCA (a secondary DNA strand
displacement primer is also used if exponential RCA is performed).
The disclosed method can be performed using any analyte. Preferred
analytes are proteins, peptides, nucleic acids, including amplified
nucleic acids such as TS-DNA and amplification target circles,
antigens and ligands. Target molecules for the disclosed method are
generally referred to herein as analytes.
[0041] The amplification target circle is released from the
reporter binding molecule prior to or during amplification. Such
release, referred to herein as decoupling, can be accomplished in
any suitable manner. In general, the manner in which the
amplification target circle is associated with, or linked or
coupled to, the reporter binding molecule determines the form of
decoupling. For example, where the amplification target circle is
base paired to a circle capture probe in the reporter binding
molecule, the amplification target circle can be decoupled from the
reporter binding molecule by disrupting the base pairing. Where the
amplification target circle is covalently coupled to the reporter
binding molecule via circle linker having a cleavable bond, the
amplification target circle can be decoupled from the reporter
binding molecule by cleaving the cleavable bond. To identify
analytes using the amplification target circles, reporter binding
molecules that are not associated with analytes should be removed
prior to decoupling.
[0042] Following decoupling, the amplification target circle can be
replicated by rolling circle amplification. Exponential rolling
circle amplification (ERCA) is the preferred form of RCA for this
purpose. If multiple different analytes are to be detected, the
amplification products of amplification target circles associated
with different analytes should be distinguishable. This can be
accomplished in any suitable manner. For example, the amplification
target circles can be in separate locations prior to decoupling and
remain separated following decoupling. The separate locations could
be determined, for example, by the location of the analytes with
which the amplification target circles are associated. In this
case, some or all of the amplification target circles can be the
same (thus producing the same amplification product). The different
locations of the amplification products identifies the analyte
involved. As another example, some or all of the amplification
target circles that are associated with different analytes can be
different (thus producing different amplification products). The
different amplification products identify the analytes involved.
Even if the amplification target circles are mixed together and/or
amplified in the same reaction, the different amplification target
circles (and thus the different corresponding analytes) can be
detected and distinguished based on the differences in the
amplification products.
[0043] The amplification products of RCA can be detected using any
suitable technique. Real time detection, that is, detection during
the RCA reaction is a preferred mode of detection with the
disclosed method. Real time detection can be facilitated by use of
Amplifluor.TM. primers. Amplifluor.TM. primers produce a
fluorescent signal when they become incorporated into a replicated
strand and are based paired with a complementary strand.
[0044] Although RCA reactions can be carried out with either linear
or geometric kinetics (Lizardi et al., 1998), the disclosed method
preferably uses geometric RCA. This latter form of RCA is referred
to as exponential rolling circle amplification (ERCA). In
exponential RCA, a secondary DNA strand displacement primer primes
replication of TS-DNA to form a complementary strand referred to as
secondary tandem sequence DNA or TS-DNA-2. As a secondary DNA
strand displacement primer is elongated, the DNA polymerase will
run into the 5' end of the next hybridized secondary DNA strand
displacement molecule and will displace its 5' end. In this fashion
a tandem queue of elongating DNA polymerases is formed on the
TS-DNA template. As long as the rolling circle reaction continues,
new secondary DNA strand displacement primers and new DNA
polymerases are added to TS-DNA at the growing end of the rolling
circle. A tertiary DNA strand displacement primer strand (which is
complementary to the TS-DNA-2 strand and which can be the rolling
circle replication primer) can then hybridize to, and prime
replication of, TS-DNA-2 to form TS-DNA-3 (which is equivalent to
the original TS-DNA). Strand displacement of TS-DNA-3 by the
adjacent, growing TS-DNA-3 strands makes TS-DNA-3 available for
hybridization with secondary DNA strand displacement primer. This
results in another round of replication resulting in TS-DNA-4
(which is equivalent to TS-DNA-2). TS-DNA-4, in turn, becomes a
template for DNA replication primed by tertiary DNA strand
displacement primer. The cascade continues this manner until the
reaction stops or reagents become limiting. The additional forms of
tandem sequence DNA beyond secondary tandem sequence DNA are
collectively referred to herein as higher order tandem sequence
DNA. Higher order tandem sequence DNA encompasses TS-DNA-3,
TS-DNA-4, and any other tandem sequence DNA produced from
replication of secondary tandem sequence DNA or the products of
such replication. In a preferred mode of ERCA, the rolling circle
replication primer serves as the tertiary DNA strand displacement
primer, thus eliminating the need for a separate primer.
[0045] The disclosed method is preferably used to detect and
analyze proteins and peptides. In preferred embodiments, multiple
proteins can be analyzed using solid supports to which the various
proteins are immobilized (if they are present in the sample being
tested). An amplification target circle is then associated with the
various proteins using a conjugate of the circle and a specific
binding molecule, such as an antibody, that is specific for the
protein to be detected. Rolling circle replication of the
amplification target circles results in production of a large
amount of DNA. The amplified DNA serves as a readily detectable
signal for the proteins. Different proteins can be distinguished in
several ways. For example, each different protein can be associated
with a different amplification target circle that in turn is
replicated to produce amplified DNA. The result is distinctive
amplified DNA for each different protein. The different amplified
DNAs can be distinguished using any suitable sequence-based nucleic
acid detection technique. In this form of the method, many proteins
can be detected in the same amplification reaction. Different
amplification target circles associated with different proteins
produce distinguishable amplified DNA which identifies the
corresponding proteins (that is, the proteins with which the
reporter binding molecules had been associated). Alternatively, the
location of the amplified DNA can indicate the protein involved if
different proteins are immobilized at predetermined locations on a
solid support.
[0046] Another embodiment of the disclosed method involves
comparison of the proteins expressed in two or more different
samples. The information generated is analogous to the type of
information gathered in nucleic acid expression profiles. For
example, the same analyte(s) from different samples can be
associated with different amplification target circles which are
replicated to produce different amplified DNAs. In this way, an
analyte from one sample will produce a different amplified DNA from
the same analyte in a different sample.
[0047] This sample-specific detection can be achieved even when the
samples are mixed together following association of the
amplification target circles with the analytes (a preferred mode of
the method). For example, different analyte capture agents can be
mixed with first and second samples, respectively. This associates
a different hapten with the same type of analyte in the different
samples. In preferred embodiments, the samples are mixed together.
The analytes can be captured on substrate, reporter binding
molecules can be associated with the analyte capture agents, and
DNA from the amplification target circles. Even if analytes from
different samples are captured at the same location on the
substrate (a preferred mode of the method), the source and amount
of each analyte present at that location can be determined by
virtue of the different amplified DNAs that will be produced.
[0048] The source of an analyte (that is, the sample from which the
analyte came) can be determined, for example, by using different
labels for different amplified DNAs (which resulted from
amplification target circles keyed to the different samples). By
using labels that can be distinguished when detected simultaneously
with other labels (such as fluorescent labels with distinct
emission spectra), all of the samples can be mixed together and
analyzed together. The detected label identifies the source of the
analyte indirectly through the chain of components: label to
amplified DNA to circular DNA to analyte.
[0049] In another form of the disclosed method, referred to as
ImmunoRCA, the amplification target circle is attached to an
antibody. In one preferred form of the disclosed method, the
antibody is directed against a hapten. In another preferred form of
the disclosed method, the antibody is directed against the analyte
itself. In the presence of a primer (referred to as a rolling
circle replication primer), DNA polymerase, and nucleotides, the
rolling circle reaction results in a DNA molecule consisting of
multiple copies of the circle DNA sequence (referred to as tandem
sequence DNA). A secondary DNA strand displacement primer is also
used if exponential RCA is performed. The amplified DNA can be
detected in a variety of ways, including direct incorporation of
hapten- or fluorescently-labeled nucleotides, or by hybridization
of fluor or enzymatically labeled complementary oligonucleotide
probes.
[0050] In another aspect, the disclosed method involves
immobilization of analytes present in complex biological samples
and determining and quantitating their presence in the samples. For
example, antigens present in biological extracts and fluids can be
identified by first selectively immobilizing them on solid
supports. An immunoRCA assay can then be employed for detection and
quantitation.
[0051] In another aspect, the disclosed method involves multiplexed
detection and quantitation of more than one analyte in a sample.
For example, a solid support can be incubated with sample
containing a mixture of protein analytes to be detected, where the
solid support contains immobilized capture antibodies (analyte
capture agents). The solid support next can be incubated with a
mixture containing at least one biotinylated antibody for each
analyte. An immunoRCA microarray assay then can be employed for
detection and quantitation.
[0052] In another aspect, an immunoRCA assay can be performed in
microwell-glass slides, where each well is separated by a Teflon
mask, or microtiter dishes. Each of the wells can be used to assay
different analytes and/or different samples, and controls.
Multiwell slides also can be printed with arrays of anti-IgE
capture antibodies in the wells. Semi-automation of immunoRCA
assays in such multiwell formats can be implemented, for example,
on inexpensive liquid handling robots.
[0053] ImmunoRCA assay can be applied to other multiplexed antibody
assays. For example, certain immunological reactions are caused by
specific IgG.sub.4 rather than IgE (AAAI Board of Directors, J
Allergy Clin Immunol. 95:652-654 (1995)). The use of an anti-human
IgG.sub.4 conjugated to a DNA circle that is different in sequence
from the DNA circle conjugated to an anti-IgE would allow the
simultaneous measurement of allergen-specific IgG.sub.4 and IgE.
Such an assay can be used during allergen desensitisation therapy
or for monitoring response to anti-IgE therapy (Chang Nature
Biotech. 18:157-162 (2000)).
[0054] The enormous multiplexing capabilities of immunoRCA, such as
the ability to detect and differentiate multiple analytes based on
the sequence of amplified DNA, can be used for clinical diagnostic
tests involving detection of multiple specific antibodies, such as
autoantibodies in suspected systemic autoimmune disorders,
inflammatory arthritis, organ-specific autoimmune disorders or,
indeed, in histocompatibility testing. Additional applications
include infectious disease diagnostics with measurement of strain-
and species-specific IgM and IgG, as well as in vitro testing of
functional antibody responses in patients with suspected primary
and secondary immunodeficiency diseases. Finally, the multiplexing,
automation and ultrasensitivity of this format can be applied to
other immunoassays besides those involving antibody detection.
RCA-powered sandwich immunoassays can provide a 8- to 9-log gain in
sensitivity (signal) over conventional assays for analytes such as
prostate serum antigen. Thus, the disclosed method produces a huge
gain in diagnostic and prognostic power made possible by the
simultaneous testing of multiple analytes for the molecular staging
of disease.
[0055] Nucleic acids are ideal molecular labels for multiple
analyte detection because different specific sequences can be
arbitrarily associated with each individual analyte. Direct
covalent coupling of nucleic acid (as a circle capture probe) to
antibody permits an unlimited number of antibody-nucleic acid
adducts to be prepared and used in any combination, provided that
each nucleic acid is unique (Hendrickson et al., Nucleic Acids Res.
23: 522-529 (1995)).
[0056] Materials
[0057] A. Analytes
[0058] The disclosed method involves the detection of analytes. In
general, any compound, moiety, or component of a compound or
complex can be an analyte. Preferred analytes are peptides,
proteins, and other macromolecules such as lipids, complex
carbohydrates, proteolipids, membrane fragments, and nucleic acids.
Analytes can also be smaller molecules such as cofactors,
metabolites, enzyme substrates, metal ions, and metal chelates.
Analytes preferably range in size from 100 daltons to 1,000,000
daltons.
[0059] Analytes may contain modifications, both naturally occurring
or induced in vitro or in vivo. Induced modifications include
adduct formation such as hapten attachment, multimerization,
complex formation by interaction with other chemical moieties,
digestion or cleavage (by, for example, protease), and metal ion
attachment or removal. The disclosed method can be used to detect
differences in the modification state of an analyte, such as the
phosphorylation or glycosylation state of proteins.
[0060] Analytes can be associated directly or indirectly with
substrates (solid supports), preferably solid supports with
multiple reaction chamers. Most preferred are microtiter dishes.
Analytes can be captured and/or immobilized using analyte capture
agents. Immobilized analytes can be used to capture other
components used in the disclosed method such as analyte capture
agents and reporter binding molecules.
[0061] B. Reporter Binding Molecules
[0062] A reporter binding molecule comprises a specific binding
molecule coupled or tethered to, or associated with, an
amplification target circle. A reporter binding molecule can also
comprise a circle capture probe, a circle linker, or both. The
specific binding molecule is referred to as the affinity portion of
the reporter binding molecule and the amplification target circle
is referred to as the nucleic acid portion of the reporter binding
molecule. The sequence of the amplification target circle sequence
can be arbitrarily chosen. In a multiplex assay using multiple
reporter binding molecules, it is preferred that the amplification
target circle sequence for each reporter binding molecule be
substantially different to limit the possibility of non-specific
target detection. Alternatively, it may be desirable in some
multiplex assays, to use amplification target circle sequences with
related sequences. Such assays can use one or a few ATCs to detect
a larger number of analytes.
[0063] Amplification target circles can be coupled or tethered to,
or associated with, a specific binding molecules in any manner that
allows release (decoupling) of the amplification target circles
from the reporter binding molecules. For example, the amplification
target circle can be base paired to a circle capture probe in the
reporter binding molecule or covalently coupled to the reporter
binding molecule via circle linker having a cleavable bond. As used
herein, decoupling refers to physical disunion of one molecule or
component from another (as for example, decoupling of an
amplification target circle from a reporter binding molecule). It
is specifically contemplated that decoupling refers to the physical
disunion both of molecules or components that are covalent couple
to each other and molecules or components that are non-covalently
associated with each other. In the former case, decoupling will
generally involve cleavage of one of more covalent bonds. In the
latter case, decoupling will generally involve dissociation. In the
case of an amplification target molecule that is tethered to a
specific binding molecule, decoupling can involve dissociation,
cleavage of one or more covalent bonds, or both.
[0064] A circle capture probe is an oligomer, such as an
oligonucleotide, that can base pair with an amplification target
circle. The region of the circle capture probe that base pairs with
the amplification target circle can be any length that supports
specific and stable hybridization between the circle capture probe
and the amplification target circle. Generally this is 12 to 100
nucleotides long, but is preferably 20 to 45 nucleotides long. The
amplification target circle can be decoupled from the reporter
binding molecule by disrupting the base pairing. In general, the
circle capture probe should be incapable of priming nucleic acid
synthesis. This can be accomplished in any suitable manner. For
example, the circle capture probe can be coupled to the specific
binding molecule via the 3' end of the circle capture probe, thus
making it unavailable for extension. The 3' end of the circle
capture probe can also be blocked to prevent extension. This can be
accomplished by, for example, modification of the 3' end
nucleotide. For example, a chemical group or molecule can be added
to the 3' end. The circle capture probe can also be composed of
subunits that do not support priming.
[0065] A circle linker is a component of a reporter molecule that
links the amplification target circle to the specific binding
molecule in a reporter binding molecule. Circle linkers preferably
have a cleavable bond. As used herein, a cleavable bond is a
covalent bond that can be easily and/or specifically cleaved. A
cleavable bond in a circle linker is used to decouple the
amplification target molecule from the reporter binding molecule.
The amplification target circle can be decoupled from the reporter
binding molecule by cleaving the cleavable bond.
[0066] Examples of useful circle linkers include linkers comprising
a disulfide bond or a dihydroxy bond. Useful examples of linkers
comprising disulfide bonds include dithiobis succinimidyl
propionate, dimethyl 3,3'-dithiobispropionimidate,
dithio-bis-maleimidoethane, 3,3'-dithiobis sulfosuccinimidyl
propionate, succinimidyl 6-[3-(2pyridyldithio)-propiona-
mido]hexonate, or N-succinimidyl 3-[2- pyridyldithio]propionate.
Useful examples of linkers comprising dihydroxy bonds include 1,4
bis-maleimidyl-2,3-dihydroxybutane, disuccinimidyl tartrate, or
disulfosuccinimidyl tartrate. Disulfide bonds can be cleaved by,
for example, treatment with a reducing agent such as
.beta.-mercaptoethanol or dithiothreitol. Dihydroxy bonds can be
cleaved by, for example, treatment with periodate. Circle linkers
can be attached to amplification target circles via a reactive
group on the amplification target circle. Numerous reactive groups
are known and can be used for this purpose. For example, the
reactive group can be an allyl amino group.
[0067] Amplification target circles can be associated with or
linked to specific binding molecules to form reporter binding
agents before, during, or after association of the specific binding
molecule with an analyte. For example, where a specific binding
molecule is coupled to a circle capture probe, the amplification
target circle can be base paired with the circle capture probe
after the specific binding molecule is associated with the analyte.
This is illustrated in FIG. 5 and Example 3. Alternatively, the
amplification target circle is base paired with the circle capture
probe before the specific binding molecule is associated with the
analyte. This is illustrated in FIG. 3 and Example 2.
[0068] Generally, an amplification target circle will be linked to
a specific binding molecule through covalent coupling. That is, the
specific binding molecule is covalently coupled to the circle
linker, and the circle linker is covalently coupled to the
amplification target circle. However, amplification target circles
can also be linked to a specific binding molecule by tethering. In
such a case the circle linker is the tether and is referred to as a
tether circle linker. An amplification target circle is tethered to
a specific binding molecule when circle linker is looped through
the amplification target circle and where both sides of the circle
linker (preferably both ends) are covalently coupled to the
specific binding molecule. Topologically, the amplification target
circle can rotate through the looped circle linker. The tether
circle linker can be any material that can form a loop and be
coupled to a specific binding molecule. Linear polymers are a
preferred material for tether circle linkers. When the cleavable
bond in the circle linker is cleaved, the tether is broken and the
amplification target circle is decoupled from the reporter binding
molecule.
[0069] As used herein, a specific binding molecule is a molecule
that interacts specifically with a particular molecule or moiety.
The molecule or moiety that interacts specifically with a specific
binding molecule can be an analyte or another molecule that serves
as an intermediate in the interaction between the specific binding
molecule and the analyte. A preferred example of such an
intermediate is an analyte capture agent. It is to be understood
that the term analyte refers to both separate molecules and to
portions of molecules, such as an epitope of a protein, that
interacts specifically with a specific binding molecule.
Antibodies, either member of a receptor/ligand pair, and other
molecules with specific binding affinities are examples of specific
binding molecules, useful as the affinity portion of a reporter
binding molecule. A reporter binding molecule with an affinity
portion that is an antibody is also referred to herein as a
reporter antibody. By coupling an amplification target circle to
such specific binding molecules, binding of a specific binding
molecule to its specific target can be detected by amplifying an
ATC with rolling circle amplification. This amplification allows
sensitive detection of a very small number of bound analytes.
[0070] A reporter binding molecule that interacts specifically with
a particular analyte is said to be specific for that analyte. For
example, a reporter binding molecule with an affinity portion that
is an antibody that binds to a particular antigen is said to be
specific for that antigen. The antigen is the analyte.
[0071] Antibodies useful as the affinity portion of reporter
binding molecules, can be obtained commercially or produced using
well established methods. For example, Johnstone and Thorpe, on
pages 30-85, describe general methods useful for producing both
polyclonal and monoclonal antibodies. The entire book describes
many general techniques and principles for the use of antibodies in
assay systems.
[0072] In use, the reporter binding molecules need not be
absolutely pure. The reporter binding molecules preferably are at
least 20% pure, more preferably at least 50% pure, more preferably
at least 80% pure, and more preferably at least 90% pure.
[0073] C. Amplification Target Circles
[0074] An amplification target circle (ATC) is a circular
single-stranded DNA molecule, generally containing between 40 to
1000 nucleotides, preferably between about 50 to 150 nucleotides,
and most preferably between about 50 to 100 nucleotides. Portions
of ATCs have specific functions making the ATC useful for rolling
circle amplification (RCA). These portions are referred to as the
primer complement portion, the detection tag portions, the
secondary target sequence portions, the address tag portions, and
the promoter portion. The primer complement portion is a required
element of an amplification target circle. Detection tag portions,
secondary target sequence portions, address tag portions, and
promoter portions are optional. Generally, an amplification target
circle is a single-stranded, circular DNA molecule comprising a
primer complement portion. Those segments of the ATC that do not
correspond to a specific portion of the ATC can be arbitrarily
chosen sequences. It is preferred that ATCs do not have any
sequences that are self-complementary. It is considered that this
condition is met if there are no complementary regions greater than
six nucleotides long without a mismatch or gap. It is also
preferred that ATCs containing a promoter portion do not have any
sequences that resemble a transcription terminator, such as a run
of eight or more thymidine nucleotides.
[0075] An amplification target circle, when replicated, gives rise
to a long DNA molecule containing multiple repeats of sequences
complementary to the amplification target circle. This long DNA
molecule is referred to herein as tandem sequences DNA (TS-DNA).
TS-DNA contains sequences complementary to the primer complement
portion and, if present on the amplification target circle, the
detection tag portions, the secondary target sequence portions, the
address tag portions, and the promoter portion. These sequences in
the TS-DNA are referred to as primer sequences (which match the
sequence of the rolling circle replication primer), spacer
sequences (complementary to the spacer region), detection tags,
secondary target sequences, address tags, and promoter sequences.
Amplification target circles are useful as components of reporter
binding molecules.
[0076] D. Rolling Circle Replication Primer
[0077] A rolling circle replication primer (RCRP) is an
oligonucleotide having sequence complementary to the primer
complement portion of an ATC. This sequence is referred to as the
complementary portion of the RCRP. The complementary portion of a
RCRP and the cognate primer complement portion can have any desired
sequence so long as they are complementary to each other. In
general, the sequence of the RCRP can be chosen such that it is not
significantly complementary to any other portion of the ATC. The
complementary portion of a rolling circle replication primer can be
any length that supports specific and stable hybridization between
the primer and the primer complement portion. Generally this is 12
to 100 nucleotides long, but is preferably 20 to 45 nucleotides
long.
[0078] It is preferred that rolling circle replication primers also
contain additional sequence at the 5' end of the RCRP that is not
complementary to any part of the ATC. This sequence is referred to
as the non-complementary portion of the RCRP. The non-complementary
portion of the RCRP, if present, serves to facilitate strand
displacement during DNA replication. The non-complementary portion
of a RCRP may be any length, but is generally 1 to 100 nucleotides
long, and preferably 4 to 8 nucleotides long. A rolling circle
replication primer can be used as the tertiary DNA strand
displacement primer in exponential rolling circle amplification.
For exponential rolling circle amplification, the sequence of the
rolling circle replication primer can be chosen such that it is not
significantly complementary to the sequence of the secondary DNA
strand displacement primer.
[0079] In preferred embodiments, rolling circle replication primers
(and other primers used in the method) can contain a spacer. The
spacer can help to overcome steric factors from the surface when
immobilized, aid in anchoring polymerase on primers, or provide
other advantages, such as control or alteration of the
hydrophobicity of elements attached to a solid support. Spacers
useful for the disclosed method include nucleotide spacers such as
poly dT or poly dA; aliphatic linkers such as C18, C12, or
multimers thereof; aromatic spacers, or RNA, DNA, PNA or
combinations thereof.
[0080] Rolling circle replication primers are preferably
Amplifluor.TM. primers. Amplifluors.TM. are fluorescent moieties
and quenchers incorporated into primers containing stem structures
(usually in hairpin or stem and loop structures) such that the
quencher moiety is in proximity with the fluorescent moiety. That
is, the quencher and fluorescent are incorporated into opposite
strands of the stem structure. In the structured state, the
quencher prevents or limits fluorescence of the fluorescent moiety.
When the stem of the primer is disrupted, the quencher and
fluorescent moiety are no longer in proximity and the fluorescent
moiety produces a fluorescent signal. In the disclosed method, use
of Amplifluor.TM. primers in ERCA produces double stranded tandem
sequence DNA where the primer stem is disrupted in favor of a
complementary, replicated strand. From a reaction initially
containing structured (that is, non-fluorescent) Amplifluor.TM.
primers, fluorescence signal increases as amplification takes
place, as more and more of the Amplifluor.TM. primers are
incorporated into double stranded TS-DNA, as the Amplifluor.TM.
stems are disrupted, and as the fluorescent moieties as
consequently unquenched. Thus, use of Amplifluor.TM. primers is
particularly suited for real-time detection of amplification in
ERCA. Amplifluor.TM. primers are also referred to herein as
fluorescent quenched primers. Thus, an Amplifluor.TM. rolling
circle replication primer is also referred to as a fluorescent
quenched rolling circle replication primer.
[0081] E. Analyte Capture Agents
[0082] An analyte capture agent is any compound that can interact
with an analyte and allow the analyte to be immobilized or
separated from other compounds and analytes. An analyte capture
agent includes an analyte interaction portion. Analyte capture
agents can also include a capture portion. Analyte capture agents
without a capture portion preferably are immobilized on a solid
support. The analyte interaction portion of an analyte capture
agent is a molecule that interacts specifically with a particular
molecule or moiety. The molecule or moiety that interacts
specifically with an analyte interaction portion can be an analyte
or another molecule that serves as an intermediate in the
interaction between the analyte interaction portion and the
analyte. It is to be understood that the term analyte refers to
both separate molecules and to portions of molecules, such as an
epitope of a protein, that interacts specifically with an analyte
interaction portion. Antibodies, either member of a receptor/ligand
pair, and other molecules with specific binding affinities are
examples of molecules that can be used as an analyte interaction
portion of an analyte capture agent. The analyte interaction
portion of an analyte capture agent can also be any compound or
composition with which an analyte can interact, such as peptides.
An analyte capture agent that interacts specifically with a
particular analyte is said to be specific for that analyte. For
example, an analyte capture agent with an analyte interaction
portion that is an antibody that binds to a particular antigen is
said to be specific for that antigen. The antigen is the
analyte.
[0083] Examples of molecules useful as the analyte interaction
portion of analyte capture agents are antibodies, such as crude
(serum) antibodies, purified antibodies, monoclonal antibodies,
polyclonal antibodies, synthetic antibodies, antibody fragments
(for example, Fab fragments); antibody interacting agents, such as
protein A, carbohydrate binding proteins, and other interactants;
protein interactants (for example avidin and its derivatives);
peptides; and small chemical entities, such as enzyme substrates,
cofactors, metal ions/chelates, and haptens. Antibodies may be
modified or chemically treated to optimize binding to surfaces
and/or targets.
[0084] Antibodies useful as the analyte interaction portion of
analyte capture agents, can be obtained commercially or produced
using well-established methods. For example, Johnstone and Thorpe,
on pages 30-85, describe general methods useful for producing both
polyclonal and monoclonal antibodies. The entire book describes
many general techniques and principles for the use of antibodies in
assay systems.
[0085] The capture portion of an analyte capture agent is any
compound that can be associated with another compound. Preferably,
a capture portion is a compound, such as a ligand or hapten, that
binds to or interacts with another compound, such as ligand-binding
molecule or an antibody. It is also preferred that such interaction
between the capture portion and the capturing component be a
specific interaction, such as between a hapten and an antibody or a
ligand and a ligand-binding molecule. Examples of haptens include
biotin, FITC, digoxigenin, and dinitrophenol. The capture portion
can be used to separate compounds or complexes associated with the
analyte capture agent from those that do not.
[0086] Capturing analytes or analyte capture agents on a substrate
may be accomplished in several ways. In one embodiment, capture
docks are adhered or coupled to the substrate. Capture docks are
compounds or moieties that mediate adherence of an analyte by
binding to, or interacting with, the capture portion on an analyte
capture agent (with which the analyte is, or will be, associated).
Capture docks immobilized on a substrate allow capture of the
analyte on the substrate. Such capture provides a convenient means
of washing away reaction components that might interfere with
subsequent steps. Alternatively, analyte capture agents can be
directly immobilized on a substrate. In this case, the analyte
capture agent need not have a capture portion.
[0087] In one embodiment, the analyte capture agent or capture dock
to be immobilized is an anti-hybrid antibody. Methods for
immobilizing antibodies and other proteins to substrates are well
established. Immobilization can be accomplished by attachment, for
example, to aminated surfaces, carboxylated surfaces or
hydroxylated surfaces using standard immobilization chemistries.
Examples of attachment agents are cyanogen bromide, succinimide,
aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable
agents, epoxides and maleimides. A preferred attachment agent is a
heterobifunctional cross-linking agent such as
N-[.gamma.-maleimidobutyryloxy]succinimide ester (GMBS). These and
other attachment agents, as well as methods for their use in
attachment, are described in Protein immobilization: fundamentals
and applications, Richard F. Taylor, ed. (M. Dekker, New York,
1991), Johnstone and Thorpe, Immunochemistry In Practice (Blackwell
Scientific Publications, Oxford, England, 1987) pages 209-216 and
241-242, and Immobilized Affinity Ligands, Craig T. Hermanson et
al., eds. (Academic Press, New York, 1992). Antibodies can be
attached to a substrate by chemically cross-linking a free amino
group on the antibody to reactive side groups present within the
substrate. For example, antibodies may be chemically cross-linked
to a substrate that contains free amino, carboxyl, or sulfur groups
using glutaraldehyde, carbodiimides, or heterobifunctional agents
such as GMBS as cross-linkers. In this method, aqueous solutions
containing free antibodies are incubated with the solid support in
the presence of glutaraldehyde or carbodiimide. For crosslinking
with glutaraldehyde the reactants can be incubated with 2%
glutaraldehyde by volume in a buffered solution such as 0.1 M
sodium cacodylate at pH 7.4. Other standard immobilization
chemistries are known by those of skill in the art.
[0088] One useful form of analyte capture agents are peptides. When
various peptides are immobilized on a solid support, they can be
used as "bait" for analytes. For example, a set of different
peptides on a solid support can be used to access whether a sample
has analytes that interact with any of the peptides. Comparisons of
different samples can be made by, for example, noting differences
in the peptides to which analytes in the different samples become
associated. In another form of the disclosed method, a set of
analyte capture agents specific for analytes of interest can be
used to access the presence of a whole suite of analytes in a
sample.
[0089] In use, the analyte capture agents need not be absolutely
pure. The analyte capture agents preferably are at least 20% pure,
more preferably at least 50% pure, more preferably at least 80%
pure, and more preferably at least 90% pure.
[0090] F. Accessory Molecules
[0091] Accessory molecules are molecules that affect the
interaction of analytes and specific binding molecules or analyte
capture agents. For example, accessory molecules can be molecules
that compete with the binding of an analyte with an analyte capture
agent or specific binding molecule. One form of competitive
accessory molecules are analogs of analytes. An analog is a
molecule that is similar in structure but different in competition.
In this context, the analyte analog should be sufficiently similar
to interact with an analyte capture agent or specific binding
molecule specific for that analyte. Accessory molecules can also be
molecules that aid or are necessary for interaction of an analyte
and a specific binding molecule or analyte capture agent. Such
accessory molecules are referred to herein as analyte binding
co-factors.
[0092] In one form of the disclosed method, accessory molecules can
be compounds that are to be tested for their effect on analyte
binding. For example, the disclosed method can be used to screen
for competitors (or binding co-factors) of an analyte interaction
with a specific binding molecule or analyte capture agent. If an
accessory molecule affects interaction of the analyte, the results
of RCA will change since the association of the reporter binding
molecule to the analyte (or of the analyte capture agent to the
analyte) will be lost or gained.
[0093] In use, the accessory molecules need not be absolutely pure.
The accessory molecules preferably are at least 20% pure, more
preferably at least 50% pure, more preferably at least 80% pure,
and more preferably at least 90% pure.
[0094] G. Detection Labels
[0095] To aid in detection and quantitation of nucleic acids
amplified using the disclosed method, detection labels can be
directly incorporated into amplified nucleic acids or can be
coupled to detection molecules. As used herein, a detection label
is any molecule that can be associated with amplified nucleic acid,
directly or indirectly, and which results in a measurable,
detectable signal, either directly or indirectly. Many such labels
for incorporation into nucleic acids or coupling to nucleic acid
probes are known to those of skill in the art. Examples of
detection labels suitable for use in the disclosed method are
radioactive isotopes, fluorescent molecules, phosphorescent
molecules, enzymes, antibodies, and ligands.
[0096] Examples of suitable fluorescent labels include fluorescein
isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red,
nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,
rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin,
BODIPY.RTM., Cascade Blue.RTM., Oregon Green.RTM., pyrene,
lissamine, xanthenes, acridines, oxazines, phycoerythrin,
macrocyclic chelates of lanthanide ions such as quantum dye.TM.,
fluorescent energy transfer dyes, such as thiazole orange-ethidium
heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
Examples of other specific fluorescent labels include
3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine
(5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red,
Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon
Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon
Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G,
BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate,
Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy F1,
Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor
RW Solution, Calcofluor White, Calcophor White ABT Solution,
Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,
Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin,
CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic
Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH--CH.sub.3,
Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid,
Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF,
Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced
Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2,
Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl
Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue,
Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, Leucophor PAF,
Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),
Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue,
Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF,
MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine,
Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear
Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole, Pacific Blue,
Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL,
Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine,
Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin,
Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant
Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD,
Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,
Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron
Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B,
Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene
Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can
C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R,
Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol
Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC,
Xylene Orange, and XRITC.
[0097] Preferred fluorescent labels are fluorescein
(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine
(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7. The absorption and emission maxima, respectively,
for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm),
Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703
nm) and Cy7 (755 mu; 778 nm), thus allowing their simultaneous
detection. Other examples of fluorescein dyes include
6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein
(TET), 2',4',5',7',1,4-hexachlorofluore- scein (HEX),
2',7'dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE),
2'-chloro-5'-fluoro-7',8'-fused
phenyl-1,4-dichloro-6-carboxyfluorescein (NED), and
2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
Fluorescent labels can be obtained from a variety of commercial
sources, including Amersham Pharmacia Biotech, Piscataway, N.J.;
Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland,
Ohio.
[0098] Additional labels of interest include those that provide for
signal only when the probe with which they are associated is
specifically bound to a target molecule. Such labels include
"molecular beacons" as described in Tyagi & Kramer, Nature
Biotechnology (1996) 14:303 and EP 0 070 685 B1. Other labels of
interest include those described in U.S. Pat. No. 5,563,037; WO
97/17471 and WO 97/17076.
[0099] Another useful label, related to molecular beacon
technology, are Amplifluors.TM.. Amplifluors.TM. are fluorescent
moieties and quenchers incorporated into primers containing stem
structures (usually in hairpin or stem and loop structures) such
that the quencher moiety is in proximity with the fluorescent
moiety. That is, the quencher and fluorescent are incorporated into
opposite strands of the stem structure. In the structured state,
the quencher prevents or limits fluorescence of the fluorescent
moiety. When the stem of the primer is disrupted, the quencher and
fluorescent moiety are no longer in proximity and the fluorescent
moiety produces a fluorescent signal. In the disclosed method, use
of Amplifluor.TM. primers in ERCA produces double stranded tandem
sequence DNA where the primer stem is disrupted in favor of a
complementary, replicated strand. From a reaction initially
containing structured (that is, non-fluorescent) Amplifluor.TM.
primers, fluorescence signal increases as amplification takes
place, as more and more of the Amplifluor.TM. primers are
incorporated into double stranded TS-DNA, as the Amplifluor.TM.
stems are disrupted, and as the fluorescent moieties as
consequently unquenched. Thus, use of Amplifluor.TM. is
particularly suited for real-time detection of amplification in
ERCA.
[0100] Labeled nucleotides are a preferred form of detection label
since they can be directly incorporated into the amplification
products during synthesis. Examples of detection labels that can be
incorporated into amplified nucleic acids include nucleotide
analogs such as BrdUrd (5-bromodeoxyuridine, Hoy and Schimke,
Mutation Research 290:217-230 (1993)), aminoallyldeoxyuridine
(Henegariu et al., Nature Biotechnology 18:345-348 (2000)),
5-methylcytosine (Sano et al., Biochim. Biophys. Acta 951:157-165
(1988)), bromouridine (Wansick et al., J. Cell Biology 122:283-293
(1993)) and nucleotides modified with biotin (Langer et al., Proc.
Natl. Acad. Sci. USA 78:6633 (1981)) or with suitable haptens such
as digoxygenin (Kerkhof, Anal. Biochem. 205:359-364 (1992)).
Suitable fluorescence-labeled nucleotides are
Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP
(Yu et al., Nucleic Acids Res., 22:3226-3232 (1994 )). A preferred
nucleotide analog detection label for DNA is BrdUrd
(bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma-Aldrich Co). Other
useful nucleotide analogs for incorporation of detection label into
DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate,
Sigma-Aldrich Co.), and 5-methyl-dCTP (Roche Molecular
Biochemicals). A preferred nucleotide analog for incorporation of
detection label into RNA is biotin-16-UTP
(biotin-16-uridine-5'-triphosphate, Roche Molecular Biochemicals).
Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct
labeling. Cy3.5 and Cy7 are available as avidin or anti-digoxygenin
conjugates for secondary detection of biotin- or
digoxygenin-labeled probes.
[0101] Detection labels that are incorporated into amplified
nucleic acid, such as biotin, can be subsequently detected using
sensitive methods known in the art. For example, biotin can be
detected using streptavidin-alkaline phosphatase conjugate (Tropix,
Inc.), which is bound to the biotin and subsequently detected by
chemiluminescence of suitable substrates (for example,
chemiluminescent substrate CSPD: disodium,
3-(4-methoxyspiro-[1,2,-dioxetane-3-2'-(5'-chloro)tricyclo[3.3.-
1.1.sup.3,7]decane]-4-yl) phenyl phosphate; Tropix, Inc.). Labels
can also be enzymes, such as alkaline phosphatase, soybean
peroxidase, horseradish peroxidase and polymerases, that can be
detected, for example, with chemical signal amplification or by
using a substrate to the enzyme which produces light (for example,
a chemiluminescent 1,2-dioxetane substrate) or fluorescent
signal.
[0102] Molecules that combine two or more of these detection labels
are also considered detection labels. Any of the known detection
labels can be used with the disclosed probes, tags, and method to
label and detect nucleic acid amplified using the disclosed method.
Methods for detecting and measuring signals generated by detection
labels are also known to those of skill in the art. For example,
radioactive isotopes can be detected by scintillation counting or
direct visualization; fluorescent molecules can be detected with
fluorescent spectrophotometers; phosphorescent molecules can be
detected with a spectrophotometer or directly visualized with a
camera; enzymes can be detected by detection or visualization of
the product of a reaction catalyzed by the enzyme; antibodies can
be detected by detecting a secondary detection label coupled to the
antibody. As used herein, detection molecules are molecules which
interact with amplified nucleic acid and to which one or more
detection labels are coupled.
[0103] H. Detection Probes
[0104] Detection probes are labeled oligonucleotides having
sequence complementary to detection tags on TS-DNA. The
complementary portion of a detection probe can be any length that
supports specific and stable hybridization between the detection
probe and the detection tag. For this purpose, a length of 10 to 35
nucleotides is preferred, with a complementary portion of a
detection probe 16 to 20 nucleotides long being most preferred.
Detection probes can contain any of the detection labels described
above. Preferred labels are biotin and fluorescent molecules. A
particularly preferred detection probe is a molecular beacon.
Molecular beacons are detection probes labeled with fluorescent
moieties where the fluorescent moieties fluoresce only when the
detection probe is hybridized (Tyagi and Kramer, Nature
Biotechnology 14:303-308 (1996)). The use of such probes eliminates
the need for removal of unhybridized probes prior to label
detection because the unhybridized detection probes will not
produce a signal. This is especially useful in multiplex assays.
The TS-DNA can be collapsed as described in WO 97/19193 using
collapsing detection probes. Collapsing TS-DNA is especially useful
with combinatorial multicolor coding, which is described below.
[0105] I. DNA Strand Displacement Primers
[0106] Primers used for secondary DNA strand displacement (an
example of which is exponential rolling circle amplification) are
referred to herein as DNA strand displacement primers. One form of
DNA strand displacement primer, referred to herein as a secondary
DNA strand displacement primer, is an oligonucleotide having
sequence matching part of the sequence of an ATC. This sequence is
referred to as the matching portion of the secondary DNA strand
displacement primer. This matching portion of a secondary DNA
strand displacement primer is complementary to sequences in TS-DNA.
The matching portion of a secondary DNA strand displacement primer
may be complementary to any sequence in TS-DNA. The matching
portion of a secondary DNA strand displacement primer can be any
length that supports specific and stable hybridization between the
primer and its complement. Generally this is 12 to 35 nucleotides
long, but is preferably 18 to 25 nucleotides long. In general, the
sequence of a secondary DNA strand displacement primer should be
chosen such that it is not significantly complementary to the
sequence of the rolling circle replication primer with which it is
used. Secondary DNA strand displacement primers are used with
tertiary strand displacement primers in exponential rolling circle
amplification. In general, the sequence of a secondary DNA strand
displacement primer should be chosen such that it is not
significantly complementary to the sequence of the tertiary DNA
strand displacement primer with which it is used.
[0107] Another form of DNA strand displacement primer, referred to
herein as a tertiary DNA strand displacement primer, is an
oligonucleotide having sequence complementary to part of the
sequence of an ATC. This sequence is referred to as the
complementary portion of the tertiary DNA strand displacement
primer. This complementary portion of the tertiary DNA strand
displacement primer matches sequences in TS-DNA. The complementary
portion of a tertiary DNA strand displacement primer may be
complementary to any sequence in the ATC. The complementary portion
of a tertiary DNA strand displacement primer can be any length that
supports specific and stable hybridization between the primer and
its complement. Generally this is 12 to 35 nucleotides long, but is
preferably 18 to 25 nucleotides long. In general, the sequence of a
tertiary DNA strand displacement primer should be chosen such that
it is not significantly complementary to the sequence of the
secondary DNA strand displacement primer with which it is used. A
preferred tertiary DNA strand displacement primer is a rolling
circle replication primer. In this case, the sequence of the
rolling circle replication primer should be chosen such that it is
not significantly complementary to the sequence of the secondary
DNA strand displacement primer with which it is used. DNA strand
displacement primers and their use are described in more detail in
U.S. Pat. No. 5,854,033 and WO 97/19193.
[0108] DNA strand displacement primers preferably are
Amplifluor.TM. primers. In the disclosed method, use of
Amplifluor.TM. primers in ERCA produces double stranded tandem
sequence DNA where the primer stem is disrupted in favor of a
complementary, replicated strand. From a reaction initially
containing structured (that is, non-fluorescent) Amplifluor.TM.
primers, fluorescence signal increases as amplification takes
place, as more and more of the Amplifluor.TM. primers are
incorporated into double stranded TS-DNA, as the Amplifluor.TM.
stems are disrupted, and as the fluorescent moieties as
consequently unquenched. Thus, use of Amplifluors.TM. is
particularly suited for real-time detection of amplification in
ERCA. If Amplifluor.TM. primers are used, only one of the primers
in a RCA reaction need be an Amplifluor.TM. primer. However, any or
all of the primers used can be Amplifluor.TM. primers, and any
combination of Amplifluor.TM. and non-Amplifluor.TM. primers can be
used. For example, the rolling circle replication primer can be
non-Amplifluor.TM. while the secondary DNA strand displacement
primer can be Amplifluor.TM., or the rolling circle replication
primers can be a mixture of Amplifluor.TM. and non-Amplifluor.TM.
primers. Amplifluor.TM. primers are also referred to herein as
fluorescent quenched primers. Thus, an Amplifluor.TM. DNA strand
displacement primer is also referred to as a fluorescent quenched
DNA strand displacement primer.
[0109] J. Oligonucleotide Synthesis
[0110] Rolling circle replication primers, circle capture probes,
circle linkers, detection probes, address probes, amplification
target circles, DNA strand displacement primers, and any other
oligonucleotides can be synthesized using established
oligonucleotide synthesis methods. Methods to produce or synthesize
oligonucleotides are well known in the art. Such methods can range
from standard enzymatic digestion followed by nucleotide fragment
isolation (see for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989 ) Chapters 5, 6) to purely
synthetic methods, for example, by the cyanoethyl phosphoramidite
method. Solid phase chemical synthesis of DNA fragments is
routinely performed using protected nucleoside cyanoethyl
phosphoramidites (S. L. Beaucage et al. (1981) Tetrahedron Lett.
22:1859). In this approach, the 3'-hydroxyl group of an initial
5'-protected nucleoside is first covalently attached to the polymer
support (R. C. Pless et al. (1975) Nucleic Acids Res. 2:773
(1975)). Synthesis of the oligonucleotide then proceeds by
deprotection of the 5'-hydroxyl group of the attached nucleoside,
followed by coupling of an incoming nucleoside-3'-phosphorami- dite
to the deprotected hydroxyl group (M. D. Matteucci et a. (1981) J.
Am. Chem. Soc. 103:3185). The resulting phosphite triester is
finally oxidized to a phosphorotriester to complete the
internucleotide bond (R. L. Letsinger et al. (1976) J. Am. Chem.
Soc. 9:3655). Alternatively, the synthesis of phosphorothioate
linkages can be carried out by sulfurization of the phosphite
triester. Several chemicals can be used to perform this reaction,
among them 3H-1,2-benzodithiole-3-one, 1,1dioxide (R. P. Iyer, W.
Egan, J. B. Regan, and S. L. Beaucage, J. Am. Chem. Soc., 1990,
112, 1253-1254). The steps of deprotection, coupling and oxidation
are repeated until an oligonucleotide of the desired length and
sequence is obtained. Other methods exist to generate
oligonucleotides such as the H-phosphonate method (Hall et al,
(1957) J. Chem. Soc., 3291-3296) or the phosphotriester method as
described by Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984),
(phosphotriester and phosphite-triester methods), and Narang et
al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method).
Protein nucleic acid molecules can be made using known methods such
as those described by Nielsen et al., Bioconjug. Chem. 5:3-7
(1994). Other forms of oligonucleotide synthesis are described in
U.S. Pat. No. 6,294,664 and U.S. Pat. No. 6,291,669.
[0111] Many of the oligonucleotides described herein are designed
to be complementary to certain portions of other oligonucleotides
or nucleic acids such that stable hybrids can be formed between
them via base pairing. The stability of these hybrids can be
calculated using known methods such as those described in Lesnick
and Freier, Biochemistry 34:10807-10815 (1995), McGraw et al.,
Biotechniques 8:674-678 (1990), and Rychlik et al., Nucleic Acids
Res. 18:6409-6412 (1990).
[0112] Oligonucleotides can be synthesized, for example, on a
Perseptive Biosystems 8909 Expedite Nucleic Acid Synthesis system
using standard .beta.-cyanoethyl phosphoramidite coupling chemistry
on synthesis columns (Glen Research, Sterling, Va.). Oxidation of
the newly formed phosphites can be carried out using, for example,
the sulfurizing reagent 3H-1,2-benzothiole-3-one-1,1-idoxide (Glen
Research) or the standard oxidizing reagent after the first and
second phosphoramidite addition steps. The thio-phosphitylated
oligonucleotides can be deprotected, for example, using 30%
ammonium hydroxide (3.0 ml) in water at 55.degree. C. for 16 hours,
concentrated in an OP 120 Savant Oligo Prep deprotection unit for 2
hours, and desalted with PD10 Sephadex columns using the protocol
provided by the manufacturer.
[0113] So long as their relevant function is maintained, rolling
circle replication primers, circle capture probes, circle linkers,
detection probes, address probes, amplification target circles, DNA
strand displacement primers, and any other oligonucleotides can be
made up of or include modified nucleotides (nucleotide analogs).
Many modified nucleotides are known and can be used in
oligonucleotides. A nucleotide analog is a nucleotide which
contains some type of modification to either the base, sugar, or
phosphate moieties. Modifications to the base moiety would include
natural and synthetic modifications of A, C, G, and T/U as well as
different purine or pyrimidine bases, such as uracil-5-yl,
hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. A modified base
includes but is not limited to 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
7-deazaguanine and 7-deazaadenine and 3-deazaguanine and
3-deazaadenine. Additional base modifications can be found for
example in U.S. Pat. No. 3,687,808, Englisch et al., Angewandte
Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y. S.,
Chapter 15, Antisense Research and Applications, pages 289-302,
Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain
nucleotide analogs, such as 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine can increase the stability of
duplex formation. Other modified bases are those that function as
universal bases. Universal bases include 3-nitropyrrole and
5-nitroindole. Universal bases substitute for the normal bases but
have no bias in base pairing. That is, universal bases can base
pair with any other base. Base modifications often can be combined
with for example a sugar modification, such as 2'-O-methoxyethyl,
to achieve unique properties such as increased duplex stability.
There are numerous U.S. Pat. Nos. such as 4,845,205; 5,130,302;
5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;
5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,
5,596,091; 5,614,617; and 5,681,941, which detail and describe a
range of base modifications. Each of these patents is herein
incorporated by reference.
[0114] Nucleotide analogs can also include modifications of the
sugar moiety. Modifications to the sugar moiety would include
natural modifications of the ribose and deoxyribose as well as
synthetic modifications. Sugar modifications include but are not
limited to the following modifications at the 2' position: OH; F;
O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or
O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or unsubstituted C1 to C10, alkyl or C2 to C10 alkenyl
and alkynyl. 2' sugar modifications also include but are not
limited to --O[(CH.sub.2)n O]m CH.sub.3, --O(CH.sub.2)n OCH.sub.3,
--O(CH.sub.2)n NH.sub.2, --O(CH.sub.2)n CH.sub.3, --O(CH.sub.2)n
--ONH.sub.2, and --O(CH.sub.2)nON[(CH.sub.2)n CH.sub.3)].sub.2,
where n and m are from 1 to about 10.
[0115] Other modifications at the 2' position include but are not
limited to: C1 to C10 lower alkyl, substituted lower alkyl,
alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,
Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2 CH.sub.3,
ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted
silyl, an RNA cleaving group, a reporter group, an intercalator, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having
similar properties. Similar modifications may also be made at other
positions on the sugar, particularly the 3' position of the sugar
on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides
and the 5' position of 5' terminal nucleotide. Modified sugars
would also include those that contain modifications at the bridging
ring oxygen, such as CH.sub.2 and S. Nucleotide sugar analogs may
also have sugar mimetics such as cyclobutyl moieties in place of
the pentofuranosyl sugar. There are numerous United States patents
that teach the preparation of such modified sugar structures such
as U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is
herein incorporated by reference in its entirety.
[0116] Nucleotide analogs can also be modified at the phosphate
moiety. Modified phosphate moieties include but are not limited to
those that can be modified so that the linkage between two
nucleotides contains a phosphorothioate, chiral phosphorothioate,
phosphorodithioate, phosphotriester, aminoalkylphosphotriester,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonate and chiral phosphonates, phosphinates, phosphoramidates
including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates. It is understood
that these phosphate or modified phosphate linkages between two
nucleotides can be through a 3'-5' linkage or a2'-5' linkage, and
the linkage can contain inverted polarity such as 3'-5' to 5'-3' or
2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are
also included. Numerous United States patents teach how to make and
use nucleotides containing modified phosphates and include but are
not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is
herein incorporated by reference.
[0117] It is understood that nucleotide analogs need only contain a
single modification, but may also contain multiple modifications
within one of the moieties or between different moieties.
[0118] Nucleotide substitutes are molecules having similar
functional properties to nucleotides, but which do not contain a
phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide
substitutes are molecules that will recognize and hybridize to
(base pair to) complementary nucleic acids in a Watson-Crick or
Hoogsteen manner, but which are linked together through a moiety
other than a phosphate moiety. Nucleotide substitutes are able to
conform to a double helix type structure when interacting with the
appropriate target nucleic acid.
[0119] Nucleotide substitutes are nucleotides or nucleotide analogs
that have had the phosphate moiety and/or sugar moieties replaced.
Nucleotide substitutes do not contain a standard phosphorus atom.
Substitutes for the phosphate can be for example, short chain alkyl
or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl
or cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages. These
include those having morpholino linkages (formed in part from the
sugar portion of a nucleoside); siloxane backbones; sulfide,
sulfoxide and sulfone backbones; formacetyl and thioformacetyl
backbones; methylene formacetyl and thioformacetyl backbones;
alkene containing backbones; sulfamate backbones; methyleneimino
and methylenehydrazino backbones; sulfonate and sulfonamide
backbones; amide backbones; and others having mixed N, O, S and CH2
component parts. Numerous United States patents disclose how to
make and use these types of phosphate replacements and include but
are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0120] It is also understood in a nucleotide substitute that both
the sugar and the phosphate moieties of the nucleotide can be
replaced, by for example an amide type linkage (aminoethylglycine)
(PNA). U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach how
to make and use PNA molecules, each of which is herein incorporated
by reference. (See also Nielsen et al., Science 254:1497-1500
(1991)).
[0121] Oligonucleotides can be comprised of nucleotides and can be
made up of different types of nucleotides or the same type of
nucleotides. For example, one or more of the nucleotides in an
oligonucleotide can be ribonucleotides, 2'-O-methyl
ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl
ribonucleotides; about 10% to about 50% of the nucleotides can be
ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of
ribonucleotides and 2'-O-methyl ribonucleotides; about 50% or more
of the nucleotides can be ribonucleotides, 2'-O-methyl
ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl
ribonucleotides; or all of the nucleotides are ribonucleotides,
2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and
2'-O-methyl ribonucleotides.
[0122] K. Solid Supports
[0123] Solid supports are solid-state substrates or supports with
which analytes (or other components used in the disclosed method)
can be associated. Analytes can be associated with solid supports
directly or indirectly. For example, analytes can be directly
immobilized on solid supports. Analyte capture agents and accessory
molecules can also be immobilized on solid supports. A preferred
form of solid support is a microtiter dish. Another form of solid
support is an array detector. An array detector is a solid support
to which multiple different address probes or detection molecules
have been coupled in an array, grid, or other organized
pattern.
[0124] Rolling circle amplification of decoupled amplification
target circles can be performed on solid supports having reaction
chambers. A reaction chamber is any structure in which a separate
amplification reaction can be performed. Useful reaction chambers
include wells, vessels, tubes, chambers, holes, depressions,
dimples, locations, or other structures that can support separate
reactions. Solid supports preferably comprise arrays of reaction
chambers. In connection with reaction chambers, a separate reaction
refers to a reaction where substantially no cross contamination of
reactants or products will occur between different reaction
chambers. Substantially no cross contamination refers to a level of
contamination of reactants or products below a level that would be
detected in the particular reaction or assay involved. For example,
if TS-DNA contamination from another reaction chamber would not be
detected in a given reaction chamber in a given assay (even though
it may be present), there is no substantial cross contamination of
the TS-DNA. It is understood, therefore, that reaction chambers can
comprise, for example, locations on a planar surface so long as the
reactions performed at the locations remain separate and are not
subject to mixing.
[0125] Solid-state substrates for use in solid supports can include
any solid material with which analytes can be associated, directly
or indirectly. This includes materials such as acrylamide, agarose,
cellulose, cellulose, nitrocellulose, glass, gold, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, glass, polysilicates,
polycarbonates, teflon, fluorocarbons, nylon, silicon rubber,
polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, functionalized silane, polypropylfumerate,
collagen, glycosaminoglycans, and polyamino acids. Solid-state
substrates can have any useful form including thin film, membrane,
bottles, dishes, fibers, woven fibers, shaped polymers, particles,
beads, microparticles, or a combination. Solid-state substrates and
solid supports can be porous or non-porous. A preferred form for a
solid-state substrate is a microtiter dish. The most preferred form
of microtiter dish is the standard 96-well type. In some
embodiments, a multiwell glass slide can be employed.
[0126] Different analytes, analyte capture agents, or accessory
molecules can be used together as a set. The set can be used as a
mixture of all or subsets of the analytes, analyte capture agents,
and accessory molecules used separately in separate reactions, or
immobilized on a solid support. Analytes, analyte capture agents,
and accessory molecules used separately or as mixtures can be
physically separable through, for example, association with or
immobilization on a solid support. An array includes a plurality of
analytes, analyte capture agents and/or accessory molecules
immobilized at identified or predefined locations on the solid
support. Each predefined location on the solid support generally
has one type of component (that is, all the components at that
location are the same). Alternatively, multiple types of components
can be immobilized in the same predefined location on a solid
support. Each location will have multiple copies of the given
components. The spatial separation of different components on the
solid support allows separate detection and identification of
analytes.
[0127] Although preferred, it is not required that the solid
support be a single unit or structure. The set of analytes, analyte
capture agents, or accessory molecules may be distributed over any
number of solid supports. For example, at one extreme, each probe
may be immobilized in a separate reaction tube or container, or on
separate beads or microparticles. Different modes of the disclosed
method can be performed with different components (for example,
analytes, analyte capture agents, and accessory molecules)
immobilized on a solid support.
[0128] In alternative embodiments, RCA is performed in solution,
and the products of the amplification are captured on a solid
support. For example, the decoupled amplification target circles
can be amplified together (that is, not in separate reaction
chambers) and the products captured. For example, a biotinylated
capture antibody can be added to a sample containing the analyte,
followed by a reporter binding molecule that binds to a different
location on the analyte. These components--the capture antibody and
the reporter binding molecule--can be added in any order. RCA then
can be performed to produce TS-DNA, and purified on a matrix
containing streptavidin (streptavidin beads (Dynal), for example).
The TS-DNA then can be detected or quantitated by hybridization to
a solid support containing oligonucleotide probes complementary to
the TS-DNA. Such probes are referred to herein as address probes.
By attaching different address probes to different regions of a
solid support, different RCA products can be captured at different,
and therefore diagnostic, locations on the solid support. For
example, in a microtiter plate multiplex assay, address probes
specific for up to 96 different TS-DNAs (each amplified via
different primers and ATCs) can be immobilized on a microtiter
plate, each in a different well. Capture and detection will occur
only in those probe elements on the solid support corresponding to
TS-DNAs for which the corresponding analytes were present in a
sample.
[0129] Methods for immobilization of oligonucleotides to
solid-state substrates are well established. Oligonucleotides,
including address probes and detection probes, can be coupled to
substrates using established coupling methods. For example,
suitable attachment methods are described by Pease et al., Proc.
Natl. Acad. Sci. USA 91(11):5022-5026 (1994), and Khrapko et al.,
Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method for
immobilization of 3'-amine oligonucleotides on casein-coated slides
is described by Stimpson et al., Proc. Natl. Acad. Sci. USA
92:6379-6383 (1995). A preferred method of attaching
oligonucleotides to solid-state substrates is described by Guo et
al., Nucleic Acids Res. 22:5456-5465 (1994).
[0130] Some solid supports useful in RCA assays have detection
antibodies attached to a solid-state substrate. Such antibodies can
be specific for a molecule of interest. Captured molecules of
interest can then be detected by binding of a second, reporter
antibody, followed by RCA. Such a use of antibodies in a solid
support allows RCA assays to be developed for the detection of any
molecule for which antibodies can be generated. Methods for
immobilizing antibodies to solid-state substrates are well
established. Immobilization can be accomplished by attachment, for
example, to aminated surfaces, carboxylated surfaces or
hydroxylated surfaces using standard immobilization chemistries.
Examples of attachment agents are cyanogen bromide, succinimide,
aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable
agents, epoxides and maleimides. A preferred attachment agent is
the heterobifunctional cross-linker N-[.gamma.-Maleimidobutyryloxy]
succinimide ester (GMBS). These and other attachment agents, as
well as methods for their use in attachment, are described in
Protein immobilization: fundamentals and applications, Richard F.
Taylor, ed. (M. Dekker, New York, 1991), Johnstone and Thorpe,
Immunochemistry In Practice (Blackwell Scientific Publications,
Oxford, England, 1987) pages 209-216 and 241-242, and Immobilized
Affinity Ligands, Craig T. Hermanson et al., eds. (Academic Press,
New York, 1992). Antibodies can be attached to a substrate by
chemically cross-linking a free amino group on the antibody to
reactive side groups present within the solid-state substrate. For
example, antibodies may be chemically cross-linked to a substrate
that contains free amino, carboxyl, or sulfur groups using
glutaraldehyde, carbodiimides, or GMBS, respectively, as
cross-linker agents. In this method, aqueous solutions containing
free antibodies are incubated with the solid-state substrate in the
presence of glutaraldehyde or carbodiimide.
[0131] A preferred method for attaching antibodies or other
proteins to a solid-state substrate is to functionalize the
substrate with an amino- or thiol-silane, and then to activate the
functionalized substrate with a homobifimctional cross-linker agent
such as (Bis-sulfo-succinimidyl suberate (BS.sup.3) or a
heterobifunctional cross-linker agent such as GMBS. For
cross-linking with GMBS, glass substrates are chemically
functionalized by immersing in a solution of
mercaptopropyltrimethoxysila- ne (1% vol/vol in 95% ethanol pH 5.5)
for 1 hour, rinsing in 95% ethanol and heating at 120.degree. C.
for 4 hrs. Thiol-derivatized slides are activated by immersing in a
0.5 mg/ml solution of GMBS in 1% dimethylformamide, 99% ethanol for
1 hour at room temperature. Antibodies or proteins are added
directly to the activated substrate, which are then blocked with
solutions containing agents such as 2% bovine serum albumin, and
air-dried. Other standard immobilization chemistries are known by
those of skill in the art.
[0132] Each of the components (analyte capture agents, accessory
molecules, and/or analytes) immobilized on the solid support
preferably is located in a different predefined region of the solid
support. The different locations preferably are different reaction
chambers. Each of the different predefined regions can be
physically separated from each other of the different regions. The
distance between the different predefined regions of the solid
support can be either fixed or variable. For example, in an array,
each of the components can be arranged at fixed distances from each
other, while components associated with beads will not be in a
fixed spatial relationship. In particular, the use of multiple
solid support units (for example, multiple beads) will result in
variable distances.
[0133] Components can be associated or immobilized on a solid
support at any density. Components preferably are immobilized to
the solid support at a density exceeding 400 different components
per cubic centimeter. Arrays of components can have any number of
components. For example, an array can have at least 1,000 different
components immobilized on the solid support, at least 10,000
different components immobilized on the solid support, at least
100,000 different components immobilized on the solid support, or
at least 1,000,000 different components immobilized on the solid
support.
[0134] L. DNA Polymerases
[0135] DNA polymerases useful in the disclosed method must be
capable, either alone or in combination with a compatible strand
displacement factor, perform rolling circle replication of primed
single-stranded circles. Such polymerases are referred to herein as
rolling circle DNA polymerases. It is preferred that a rolling
circle DNA polymerase lack a 5' to 3' exonuclease activity. Strand
displacement is necessary to result in synthesis of multiple tandem
copies of an amplification target circle. A 5' to 3' exonuclease
activity, if present, might result in the destruction of the
synthesized strand. It is also preferred that DNA polymerases for
use in the disclosed method are highly processive. The suitability
of a DNA polymerase for use in the disclosed method can be readily
determined by assessing its ability to carry out strand
displacement replication. Preferred strand displacement DNA
polymerases are Bst large fragment DNA polymerase (Exo(-) Bst;
Aliotta et al., Genet. Anal. (Netherlands) 12:185-195 (1996)),
exo(-)Bca DNA polymerase (Walker and Linn, Clinical Chemistry
42:1604-1608 (1996)), and bacteriophage .phi.29 DNA polymerase
(U.S. Pat. Nos. 5,198,543 and 5,001,050 to Blanco et al.). Other
useful polymerases include phage M2 DNA polymerase (Matsumoto et
al., Gene 84:247 (1989)), phage .phi.PRD1 DNA polymerase (Jung et
al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)),exo(-)VENT.RTM. DNA
polymerase (Kong et al., J. Biol. Chem. 268:1965-1975 (1993)),
Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J
Biochem. 45:623-627 (1974)), T5 DNA polymerase (Chattejee et al.,
Gene 97:13-19 (1991)), Sequenase (U.S. Biochemicals), PRD1 DNA
polymerase (Zhu and Ito, Biochim. Biophys. Acta. 1219:267-276
(1994)), and T4 DNA polymerase holoenzyme (Kaboord and Benkovic,
Curr. Biol. 5:149-157 (1995)). Bst DNA polymerase is most
preferred.
[0136] Strand displacement can be facilitated through the use of a
strand displacement factor, such as helicase. It is considered that
any DNA polymerase that can perform strand displacement replication
in the presence of a strand displacement factor is suitable for use
in the disclosed method, even if the DNA polymerase does not
perform strand displacement replication in the absence of such a
factor. Strand displacement factors useful in strand displacement
replication include BMRF1 polymerase accessory subunit (Tsurumi et
al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding
protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164
(1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J.
Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl.
Acad. Sci. USA 91(22):10665-10669 (1994)); single-stranded DNA
binding proteins (SSB; Rigler and Romano, J. Biol. Chem.
270:8910-8919 (1995)); phage T4 gene 32 protein (Villemain and
Giedroc, Biochemistry 35:14395-14404 (1996); and calf thymus
helicase (Siegel et al., J. Biol. Chem. 267:13629-13635
(1992)).
[0137] The ability of a polymerase to carry out rolling circle
replication can be determined by using the polymerase in a rolling
circle replication assay such as those described in Fire and Xu,
Proc. Natl. Acad. Sci. USA 92:4641-4645 (1995).
[0138] The materials described above can be packaged together in
any suitable combination as a kit useful for performing the
disclosed method. For example, a kit can include a plurality of
reporter binding molecules and/or a plurality of analyte capture
agents. The analyte capture agents in the kit can be associated
with a solid support.
[0139] Method
[0140] The disclosed method is a form of rolling circle
amplification (RCA) where a reporter binding molecule provides the
amplification target circle for amplification. The disclosed method
allows RCA to produce an amplified signal (that is, tandem sequence
DNA (TS-DNA)) based on association of the reporter binding molecule
with a target molecule (also referred to as an analyte). The
specific amplification target circle that is a part of the reporter
binding molecule provides the link between the specific interaction
of the reporter binding molecule to an analyte (via the affinity
portion of the reporter binding molecule) and RCA. Once the
reporter binding molecule is associated with an analyte, a rolling
circle replication primer is hybridized to the amplification target
circle (ATC) of the reporter binding molecule, followed by
amplification of the ATC by RCA (a secondary DNA strand
displacement primer is also used if exponential RCA is performed).
The disclosed method can be performed using any analyte. Preferred
analytes are proteins, peptides, nucleic acids, including amplified
nucleic acids such as TS-DNA and amplification target circles,
antigens and ligands. Target molecules for the disclosed method are
generally referred to herein as analytes.
[0141] The amplification target circle is released from the
reporter binding molecule prior to amplification. Such release,
referred to herein as decoupling, can be accomplished in any
suitable manner. In general, the manner in which the amplification
target circle is associated with, or linked or coupled to, the
reporter binding molecule determines the form of decoupling. For
example, where the amplification target circle is base paired to a
circle capture probe in the reporter binding molecule, the
amplification target circle can be decoupled from the reporter
binding molecule by disrupting the base pairing. Where the
amplification target circle is covalently coupled to the reporter
binding molecule via circle linker having a cleavable bond, the
amplification target circle can be decoupled from the reporter
binding molecule by cleaving the cleavable bond. To identify
analytes using the amplification target circles, reporter binding
molecules that are not associated with analytes should be removed
prior to decoupling.
[0142] Following decoupling, the amplification target circle can be
replicated by rolling circle amplification. If multiple different
analytes are to be detected, the amplification products of
amplification target circles associated with different analytes
should be distinguishable. This can be accomplished in any suitable
manner. For example, the amplification target circles can be in
separate locations prior to decoupling and remain separated
following decoupling. The separate locations could be determined,
for example, by the location of the analytes with which the
amplification target circles are associated. In this case, some or
all of the amplification target circles can be the same (thus
producing the same amplification product). The different locations
of the amplification products identifies the analyte involved. As
another example, some or all of the amplification target circles
that are associated with different analytes can be different (thus
producing different amplification products). The different
amplification products identify the analytes involved. Even if the
amplification target circles are mixed together and/or amplified in
the same reaction, the different amplification target circles (and
thus the different corresponding analytes) can be detected and
distinguished based on the differences in the amplification
products.
[0143] The amplification products of RCA can be detected using any
suitable technique. Real time detection, that is, detection during
the RCA reaction is a preferred mode of detection with the
disclosed method. Real time detection can be facilitated by use of
Amplifluor.TM. primers. Amplifluor.TM. primers produce a
fluorescent signal when they become incorporated into a replicated
strand and are based paired with a complementary strand.
[0144] The disclosed method is particularly useful for generating a
profile of analytes present in a given sample. For example, the
presence and amount of various proteins present in cells can be
assessed, thus providing a direct protein expression profile. Such
analysis, a form of proteomics, is analogous to genomics analysis
of the presence and expression of nucleic acids. Multiple analyte
analysis, such as the proteomics mode of the disclosed invention,
is preferably carried out using sets of analyte capture agents. By
including in the set analyte capture agents specific for all of the
analytes to be assessed, the full range of analytes can be assayed
in a single procedure. This form of the method also allows easy
comparison of the same suite of analytes in multiple samples.
[0145] In a preferred form of the disclosed method, the analytes in
two (or more) different samples can be assessed in the same
reaction by mixing a different set of reporter binding molecules
with each sample. Each set of reporter binding molecules has the
same set of specific binding molecules but a different set of
amplification target circles. By making the different amplification
target circles specific for different rolling circle replication
primers (and different secondary DNA strand displacement primers if
exponential RCA is performed), the amplification of a specific
amplification target circle will indicate in which sample the
corresponding analyte is present. Alternatively, by using different
detection tag sequences in the different amplification target
circles the amplification products of the different amplification
target circles can be distinguished. This allows the identification
of the analyte corresponding to a given amplification target
circle.
[0146] Identification of multiple analytes can be facilitated by
using analyte capture agents to capture and/or separate analytes
based on their identity. For example, a set of immobilized analyte
capture agents can be used to associate particular analytes with
predefined regions on a solid support. Detection of an analyte in
that region identifies the analyte. One useful form of analyte
capture agent is peptides. When various peptides are immobilized on
a solid support, they can be used as "bait" for analytes. For
example, an array of different peptides can be used to access
whether a sample has analytes that interact with any of the
peptides. Comparisons of different samples can be made by, for
example, noting differences in the peptides to which analytes in
the different samples become associated. In another form of the
disclosed method, a set of analyte capture agents specific for
analytes of interest can be used to access the presence of a whole
suite of analytes in a sample.
[0147] In another form of the disclosed method, accessory molecules
can be used to affect the interaction of analytes with specific
binding molecules or analyte capture agents. For example, the
disclosed method can be used to screen for competitors (or binding
co-factors) of an analyte interaction with a specific binding
molecule or analyte capture agent. If an accessory molecule affects
interaction of the analyte, the results of RCA will change since
the association of the reporter binding molecule to the analyte (or
of the analyte capture agent to the analyte) will be lost or
gained.
[0148] Different modified forms of analytes can also be detected
with the disclosed method. For example, phosphorylated and
glycosylated forms of proteins can be detected. This can be
accomplished, for example, by using reporter binding molecules
having specific binding molecules specific for the different forms
of analyte.
[0149] In another aspect, the disclosed method involves
immobilization of analytes present in complex biological samples
and determining and quantitating their presence in the samples. In
another aspect, the disclosed method involves multiplexed detection
and quantitation of more than one analytes in a sample. For
example, a solid support containing immobilized capture antibodies
can be incubated with sample containing a mixture of protein
analytes to be detected. The solid support next can be incubated
with a mixture containing at least one biotinylated antibody for
each analyte. An immunoRCA assay then can be employed for detection
and quantitation.
[0150] In another aspect, an immunoRCA assay can be performed in 16
microwell-glass slides, wherein each well is separated by a Teflon
mask. Each of these wells can be used, for example, to assay
different samples and controls, to assay different analytes, or to
assay different sets of analytes. Multiwell slides also can be
printed with arrays of anti-IgE capture antibodies in the wells.
Semi-automation of immunoRCA assays on allergen microarrays in this
multiwell format can be implemented, for example, on an inexpensive
Beckman BioMek liquid handling robot.
[0151] ImmunoRCA assay can be applied to other multiplexed antibody
assays. For example, certain immunological reactions are caused by
specific IgG.sub.4 rather than IgE (AAAI Board of Directors, J.
Allergy Clin Immunol. 95:652-654 (1995)). The use of an anti-human
IgG.sub.4 conjugated to a DNA circle that is different in sequence
from the DNA circle conjugated to an anti-IgE would allow the
simultaneous measurement of allergen-specific IgG.sub.4 and IgE.
Such an assay can be used during allergen desensitization therapy
or for monitoring response to anti-IgE therapy (Chang Nature
Biotech. 18:157-162 (2000)).
[0152] The disclosed method generally includes the following
steps:
[0153] (a) Bringing into contact one or more analyte samples and
one or more reporter binding molecules, incubating the analyte
samples and the reporter binding molecules under conditions that
promote interaction of the specific binding molecules and analytes,
and separating the specific binding molecules that interact with
the analytes from the specific binding molecules that do not
interact with the analytes. Each reporter binding molecule
comprises a specific binding molecule and an amplification target
circle, wherein each specific binding molecule interacts with an
analyte directly or indirectly.
[0154] (b) Decoupling the amplification target circles from the
reporter binding molecules that interact with the analytes.
[0155] (c) Bringing into contact the amplification target circles
and one or more rolling circle replication primers, and incubating
the rolling circle replication primers and amplification target
circles under conditions that promote hybridization between the
amplification target circles and the rolling circle replication
primers. The amplification target circles each comprise a
single-stranded, circular DNA molecule comprising a primer
complement portion, wherein the primer complement portion is
complementary to at least one of the rolling circle replication
primers.
[0156] (d) Incubating the rolling circle replication primers and
amplification target circles under conditions that promote
replication of the amplification target circles. Replication of the
amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
[0157] The method can also be performed where at least one of the
reporter binding molecules further comprises a circle capture
probe, and where the amplification target circle of the reporter
binding molecule is associated with the reporter binding molecule
via a non-covalent interaction with the circle capture probe. The
non-covalent interaction can be base pairing. Decoupling of the
amplification target circle can be accomplished by disrupting the
base pairing. Base pairing can be disrupted by heating the reporter
binding molecules. The circle capture probe can comprise an
oligonucleotide. In some embodiments, the oligonucleotide cannot be
extended. For example, the oligonucleotide can comprise a 3' end
and a 5' end, wherein only the 5' end is free. The oligonucleotide
can be coupled to the specific binding molecule of the reporter
binding molecule via the 3' end of the oligonucleotide, the 3' end
of the oligonucleotide can be blocked, or the oligonucleotide can
be blocked.
[0158] The method can also be performed where at least one of the
reporter binding molecules further comprises a circle linker, and
where the amplification target circle of the reporter binding
molecule is coupled to the reporter binding molecule via the circle
linker. The circle linker can comprise a cleavable bond. Decoupling
of the amplification target circle can be accomplished by cleaving
the cleavable bond. In some embodiments, the cleavable bond can be
cleaved by treatment with a reducing agent. The cleavable bond can
be a disulfide bond. For example the circle linker can comprise
dithiobis succinimidyl propionate, dimethyl
3,3'-dithiobispropionimidate, dithio-bis-maleimidoethane,
3,3'-dithiobis sulfosuccinimidyl propionate, succinimidyl
6-[3-(2-pyridyldithio)-propionamido]hexonate, or N-succinimidyl
3-[2-pyridyldithio]propionate. In some embodiments, the cleavable
bond can be cleaved by treatment with periodate. The cleavable bond
can be a dihydroxy bond. For example, the circle linker can
comprise 1,4 bis-maleimidyl-2,3-dihydroxybutane, disuccinimidyl
tartrate, or disulfosuccinimidyl tartrate. The circle linker can be
coupled to the amplification target circle via a reactive group on
the amplification target circle. The reactive group can be an allyl
amino group.
[0159] The method can be performed wherein a plurality of reporter
binding molecules are brought into contact with the one or more
analyte samples; wherein a plurality of analyte samples are brought
into contact with the one or more reporter binding molecules;
wherein at least one of the analytes is a protein or peptide;
wherein at least one of the analytes is a lipid, glycolipid, or
proteoglycan; wherein at least one of the analytes is from a human
source; wherein at least one of the analytes is from a non-human
source; wherein none of the analytes are nucleic acids; wherein at
least one of the specific binding molecules is an antibody specific
for at least one of the analytes; wherein at least one of the
specific binding molecules is a molecule that specifically binds to
at least one of the analytes; wherein at least one of the specific
binding molecules is a molecule that specifically binds to at least
one of the analytes in combination with an accessory molecule;
and/or wherein the specific binding molecules and analytes interact
by binding to each other directly or indirectly. The reporter
binding molecules can be at least 20% pure, at least 50% pure, at
least 80% pure, or at least 90% pure.
[0160] The method can also include bringing into contact at least
one of the analyte samples and one or more analyte capture agents,
and separating analyte capture agents from the analyte samples,
thus separating analytes from the analyte samples. Each analyte
capture agent interacts with an analyte directly or indirectly, and
at least one analyte, if present in the analyte sample, interacts
with at least one analyte capture agent. The method can also
include bringing into contact at least one of the analyte samples
and at least one of the reporter binding molecules with at least
one accessory molecule. The accessory molecule affects the
interaction of at least one of the analytes and at least one of the
specific binding molecules or at least one of the analyte capture
agents.
[0161] The method can further comprise, simultaneous with, or
following, step (d), bringing into contact a secondary DNA strand
displacement primer and the tandem sequence DNA, and incubating
under conditions that promote (i) hybridization between the tandem
sequence DNA and the secondary DNA strand displacement primer, and
(ii) replication of the tandem sequence DNA, wherein replication of
the tandem sequence DNA results in the formation of secondary
tandem sequence DNA. In this form of the method, the rolling circle
replication primer can hybridize to the secondary tandem sequence
DNA and the secondary tandem sequence DNA can be replicated to form
tertiary tandem sequence DNA. The rolling circle replication primer
and secondary DNA strand displacement primer can continue to
hybridize with and replicate the tandem sequence DNA, secondary
tandem sequence DNA, tertiary tandem sequence DNA (and other higher
order tandem sequence DNAs) to form more amplified DNA (that is,
various generations of tandem sequence DNA).
[0162] This form of the method can further comprise, simultaneous
with, or following, step (d), bringing into contact a tertiary DNA
strand displacement primer and the secondary tandem sequence DNA,
and incubating under conditions that promote (i) hybridization
between the secondary tandem sequence DNA and the tertiary DNA
strand displacement primer, and (ii) replication of the secondary
tandem sequence DNA, wherein replication of the secondary tandem
sequence DNA results in the formation of tertiary tandem sequence
DNA. The tertiary DNA strand displacement primer and secondary DNA
strand displacement primer can continue to hybridize with and
replicate the tandem sequence DNA, secondary tandem sequence DNA,
tertiary tandem sequence DNA (and other higher order tandem
sequence DNAs) to form more amplified DNA (that is, various
generations of tandem sequence DNA). In this form of the method,
the rolling circle replication primer can be used as the tertiary
DNA strand displacement primer.
[0163] The method can be performed wherein a plurality of reporter
binding molecules are brought into contact with one or more analyte
samples, wherein two or more of the amplification target circles
are replicated in the same reaction, wherein the amplification
target circles replicated in the same reaction are different,
wherein each different amplification target circle produces a
different tandem sequence DNA, wherein the presence or absence of
different analytes is indicated by the presence or absence of
corresponding tandem sequence DNA. Replication of each different
amplification target circle can be primed by a different one of the
rolling circle replication primers.
[0164] The method can be performed wherein at least one of the
analytes is associated with a solid support. The solid support can
comprise one or more reaction chambers, wherein a plurality of the
analytes associated with the solid support are associated with the
solid support in the same reaction chamber. At least one of the
analytes associated with the solid support can be associated with
the solid support indirectly. The analytes associated with the
solid support can interact with analyte capture agents, wherein the
analyte capture agents are associated with the solid support
thereby indirectly associating the analytes with the solid
support.
[0165] The method can be performed wherein at least one specific
binding molecule interacts with at least one analyte indirectly.
The analyte can interact with an analyte capture agent, wherein the
specific binding molecule interacts with the analyte capture agent
thereby indirectly associating the specific binding molecule with
the analyte. The method can be performed wherein at least one of
the analytes is a modified form of another analyte, wherein the
specific binding molecule of at least one of the reporter binding
molecules interacts, directly or indirectly, with the analyte that
is a modified form of the other analyte, wherein the specific
binding molecule of another reporter binding molecule interacts,
directly or indirectly, with the other analyte. The analytes can be
proteins, wherein the modification of the modified form of the
other analyte can be a post-translational modification. The
modification can be phosphorylation or glycosylation.
[0166] The method can be performed wherein detection of the tandem
sequence DNA is accomplished by mixing a set of detection probes
with the tandem sequence DNA under conditions that promote
hybridization between the tandem sequence DNA and the detection
probes. A plurality of different tandem sequence DNAs can be
detected separately and simultaneously via multiplex detection. The
set of detection probes can be labeled using combinatorial
multicolor coding.
[0167] In one form of the method, the specific binding molecules
that interact with the analytes can be separated by bringing into
contact at least one of the analyte samples and one or more analyte
capture agents, and separating analyte capture agents from the
analyte samples, thus separating specific binding molecules that
interact with the analytes from the analyte samples. Each analyte
capture agent can interact with an analyte directly or indirectly,
and at least one analyte, if present in the analyte sample, can
interact with at least one analyte capture agent. At least one
analyte capture agent can be associated with a solid support,
wherein analytes that interact with the analyte capture agent
associated with a solid support become associated with the solid
support. The solid support can comprise one or more reaction
chambers, wherein a plurality of the analyte capture agents are
located in the same reaction chamber on the solid support.
[0168] In this form of the method, a plurality of reporter binding
molecules can be brought into contact with one or more analyte
samples, wherein two or more of the amplification target circles
are replicated in the same reaction chamber of the solid support,
wherein the amplification target circles replicated in the same
reaction chamber of the solid support are different, and wherein
each different amplification target circle produces a different
tandem sequence DNA. The presence or absence of different analytes
is indicated by the presence or absence of corresponding tandem
sequence DNA. Replication of each different amplification target
circle can be primed by a different one of the rolling circle
replication primers.
[0169] The solid support can comprise acrylamide, agarose,
cellulose, cellulose, nitrocellulose, glass, gold, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, glass, polysilicates,
polycarbonates, teflon, fluorocarbons, nylon, silicon rubber,
polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, fimctionalized silane, polypropylfumerate,
collagen, glycosaminoglycans, or polyamino acids.
[0170] This form of the method can further comprise bringing into
contact at least one of the analyte samples and at least one of the
reporter binding molecules with at least one accessory molecule,
wherein the accessory molecule affects the interaction of at least
one of the analytes and at least one of the specific binding
molecules or at least one of the analyte capture agents. The
accessory molecule can be brought into contact with at least one of
the analyte samples, at least one of the reporter binding
molecules, or both, prior to, simultaneous with, or following step
(a). At least one analyte capture agent can be associated with a
solid support, wherein the accessory molecule is associated with
the solid support. The accessory molecule can be associated with
the solid support by bringing the accessory molecule into contact
with the solid support prior to, simultaneous with, or following
step (a). The accessory molecule can be a protein kinase, a protein
phosphatase, an enzyme, or a compound. The accessory molecule can
be a molecule of interest, wherein one or more of the analytes are
test molecules, wherein interactions of the test molecules with the
molecule of interest are detected. At least one of the analytes can
be a molecule of interest, wherein the accessory molecule is a test
molecule, wherein interactions of the test molecule with the
molecule of interest are detected.
[0171] In this form of the method, the analyte samples can include
one or more first analyte samples and one or more second analyte
samples, wherein the reporter binding molecules include one or more
first reporter binding molecules and one or more second reporter
binding molecules. The method can further comprise, following step
(a) and prior to bringing the analyte samples and the solid support
into contact, mixing one or more of the first analyte samples and
one or more of the second analyte samples. For each first reporter
binding molecule there is a matching second reporter binding
molecule, wherein the specific binding molecules of the first
reporter binding molecules interacts with the same analyte as the
specific binding molecules of the matching second reporter binding
molecule. The amplification target circle of each different
reporter binding molecule is different, and each different
amplification target circle produces a different tandem sequence
DNA. The presence or absence of the same analyte in different
analyte samples is indicated by the presence or absence of
corresponding tandem sequence DNA. Replication of each different
amplification target circle can be primed by a different one of the
rolling circle replication primers. The tandem sequence DNA
corresponding to one of the analytes and produced in association
with a first reporter binding molecule is in the same location on
the solid support as tandem sequence DNA corresponding to the same
analyte and produced in association with the matching second
reporter binding molecule. The presence or absence of the same
analyte in different analyte samples is indicated by the presence
or absence of corresponding tandem sequence DNA.
[0172] In this form of the method, at least one of the analyte
capture agents is a molecule of interest, wherein one or more of
the analytes are test molecules, wherein interactions of the test
molecules with the molecule of interest are detected; or at least
one of the analytes is a molecule of interest, wherein one or more
of the analyte capture agents are test molecules, wherein
interactions of the test molecules with the molecule of interest
are detected.
[0173] Another form of the method further comprises, prior to,
simultaneous with, or following step (a), bringing into contact one
or more first analyte capture agents and one or more first analyte
samples, and bringing into contact one or more second analyte
capture agents and one or more second analyte samples. Each analyte
capture agent comprises an analyte interaction portion and a
capture portion, wherein for each first analyte capture agent there
is a matching second analyte capture agent. The analyte interaction
portions of the first analyte capture agents interact with the same
analyte as the analyte interaction portions of the matching second
analyte capture agents. The capture portions of the first and
second analyte capture agents each interact with a specific binding
molecule of one or more of the reporter binding molecules, wherein
the capture portions of the first analyte capture agents interact
with different specific binding molecules than the capture portions
of the matching second analyte capture agents. Each different
specific binding molecule is part of a different one of the
reporter binding molecules, wherein the amplification target circle
of each different reporter binding molecule is different, wherein
replication of each different amplification target circle is primed
by a different one of the rolling circle replication primers,
wherein each different amplification target circle produces a
different tandem sequence DNA, wherein the amplification target
circle of a reporter binding molecule that comprises a specific
binding molecule that interacts with an analyte capture agent
corresponds to the analyte capture agent. The presence or absence
of the same analyte in different analyte samples is indicated by
the presence or absence of corresponding tandem sequence DNA.
[0174] This form of the method can further comprise mixing one or
more of the first analyte samples and one or more of the second
analyte samples, or mixing the one or more first analyte capture
agents and the one or more second analyte capture agents. Mixing
the one or more first analyte capture agents and the one or more
second analyte capture agents can be accomplished by associating,
simultaneously or sequentially, the one or more first analyte
capture agents and the one or more second analyte capture agents
with the same solid support.
[0175] In this form of the method, the tandem sequence DNA
corresponding to one of the analytes and produced in association
with a first analyte capture agent can be in the same location as,
and can be simultaneously detected with, tandem sequence DNA
corresponding to the same analyte and produced in association with
the matching second analyte capture agent. The presence or absence
of the same analyte in different analyte samples is indicated by
the presence or absence of corresponding tandem sequence DNA.
[0176] In this form of the method, the capture portion of each
first analyte capture agent can be the same, wherein the reporter
binding molecules corresponding to the first analyte capture agents
are the same, wherein the amplification target circles
corresponding to the first analyte capture agents are the same. The
capture portion of each second analyte capture agent can be the
same, wherein the reporter binding molecules corresponding to the
second analyte capture agents are the same, wherein the
amplification target circles corresponding to the second analyte
capture agents are the same.
[0177] In another form of the method, at least one accessory
molecule can be brought into contact with at least one of the
analyte samples and at least one of the reporter binding molecules,
wherein the accessory molecule affects the interaction of at least
one of the analytes and at least one of the specific binding
molecules or at least one of the analyte capture agents. The
accessory molecule can compete with the interaction of at least one
of the specific binding molecules or at least one of the analyte
capture agents. The accessory molecule can be an analog of at least
one of the analytes. The accessory molecule can facilitate the
interaction of at least one of the specific binding molecules or at
least one of the analyte capture agents. The accessory molecule can
be brought into contact with at least one of the analyte samples,
at least one of the reporter binding molecules, or both, prior to,
simultaneous with, or following step (a).
[0178] In this form of the method, the accessory molecule can be a
protein kinase, a protein phosphatase, an enzyme, or a compound.
The accessory molecule can be at least 20% pure, at least 50% pure,
at least 80% pure, or at least 90% pure.
[0179] Another form of the disclosed method generally includes the
following steps:
[0180] (a) Bringing into contact one or more analyte samples and
one or more analyte capture agents, and incubating the analyte
samples and the analyte capture agents under conditions that
promote interaction of the analyte capture agents and analytes.
Each analyte capture agent can interact with an analyte directly or
indirectly. At least one analyte, if present in the analyte sample,
can interact with at least one analyte capture agent.
[0181] (b) Bringing into contact at least one of the analyte
samples and one or more reporter binding molecules, incubating the
analyte samples and the reporter binding molecules under conditions
that promote interaction of the specific binding molecules and
analyte capture agents, and separating the specific binding
molecules that interact with the analyte capture agents from the
specific binding molecules that do not interact with the analyte
capture agents. Each reporter binding molecule can comprise a
specific binding molecule and an amplification target circle, and
each specific binding molecule can interact with an analyte capture
agent directly or indirectly.
[0182] (c) Decoupling the amplification target circles from the
reporter binding molecules that interact with the analyte capture
agents.
[0183] (d) Bringing into contact the amplification target circles
and one or more rolling circle replication primers, and incubating
the rolling circle replication primers and amplification target
circles under conditions that promote hybridization between the
amplification target circles and the rolling circle replication
primers. The amplification target circles each can comprise a
single-stranded, circular DNA molecule comprising a primer
complement portion, and the primer complement portion is
complementary to at least one of the rolling circle replication
primers.
[0184] (e) Incubating the rolling circle replication primers and
amplification target circles under conditions that promote
replication of the amplification target circles. Replication of the
amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
[0185] Another form of the disclosed method generally includes the
following steps:
[0186] (a) Treating one or more analyte samples so that one or more
analytes are modified.
[0187] (b) Bringing into contact at least one of the analyte
samples and one or more reporter binding molecules, incubating the
analyte samples and the reporter binding molecules under conditions
that promote interaction of the specific binding molecules and
modified analytes, and separating the specific binding molecules
that interact with the modified analytes from the specific binding
molecules that do not interact with the modified analytes. Each
reporter binding molecule can comprise a specific binding molecule
and an amplification target circle, and each specific binding
molecule can interact with a modified analyte directly or
indirectly.
[0188] (c) Decoupling the amplification target circles from the
reporter binding molecules that interact with the modified
analytes.
[0189] (d) Bringing into contact the amplification target circles
and one or more rolling circle replication primers, and incubating
the rolling circle replication primers and amplification target
circles under conditions that promote hybridization between the
amplification target circles and the rolling circle replication
primers. The amplification target circles each can comprise a
single-stranded, circular DNA molecule comprising a primer
complement portion, and the primer complement portion is
complementary to at least one of the rolling circle replication
primers.
[0190] (e) Incubating the rolling circle replication primers and
amplification target circles under conditions that promote
replication of the amplification target circles. Replication of the
amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding modified analytes.
[0191] In this form of the method, all of the analytes can be
modified by associating a modifying group to the analytes, wherein
the modifying group is the same for all of the analytes, wherein
all of the specific binding molecules interact with the modifying
group.
[0192] Another form of the disclosed method generally includes the
following steps:
[0193] (a) Bringing into contact one or more analyte samples and a
set of analyte capture agents, a set of accessory molecules, or
both. Each analyte capture agent can interact with an analyte
directly or indirectly.
[0194] (b) Prior to, simultaneous with, or following step (a),
bringing into contact at least one of the analyte samples and one
or more reporter binding molecules. Each reporter binding molecule
can comprise a specific binding molecule and an amplification
target circle, each specific binding molecule can interact with an
analyte directly or indirectly, and each accessory molecule can
affect the interaction of at least one of the analytes and at least
one of the specific binding molecules or at least one of the
analyte capture agents.
[0195] (c) Simultaneous with, or following, steps (a) and (b),
incubating the analyte samples, the analyte capture agents, the
accessory molecules, and the reporter binding molecules under
conditions that promote interaction of the specific binding
molecules, analytes, analyte capture agents, and accessory
molecules, separating the specific binding molecules that interact
with the analytes from the specific binding molecules that do not
interact with the analytes, and decoupling the amplification target
circles from the reporter binding molecules that interact with the
analytes.
[0196] (d) Bringing into contact the amplification target circles
and one or more rolling circle replication primers, and incubating
the rolling circle replication primers and amplification target
circles under conditions that promote hybridization between the
amplification target circles and the rolling circle replication
primers. The amplification target circles each can comprise a
single-stranded, circular DNA molecule comprising a primer
complement portion, and the primer complement portion is
complementary to at least one of the rolling circle replication
primers.
[0197] (e) Incubating the reporter binding molecules and
amplification target circles under conditions that promote
replication of the amplification target circles. Replication of the
amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
[0198] The method can also be performed where the analyte capture
agents are immobilized on a solid support, where the solid support
comprises one or more reaction chambers, and where a plurality of
the analyte capture agents are immobilized in the same reaction
chamber of the solid support. The analyte capture agents can be
immobilized to the solid support at a density exceeding 400
different analyte capture agents per cubic centimeter. The analyte
capture agents can be peptides. Each of the different peptides can
be at least 4 amino acids in length, from about 4 to about 20 amino
acids in length, at least 10 amino acids in length, or at least 20
amino acids in length.
[0199] The solid support can comprise a plurality of reaction
chambers. The solid support can comprise acrylamide, agarose,
cellulose, cellulose, nitrocellulose, glass, gold, polystyrene,
polyethylene vinyl acetate, polypropylene, polymethacrylate,
polyethylene, polyethylene oxide, glass, polysilicates,
polycarbonates, teflon, fluorocarbons, nylon, silicon rubber,
polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, functionalized silane, polypropylfumerate,
collagen, glycosaminoglycans, or polyamino acids. The analyte
capture agents in the reaction chambers can be at least 20% pure,
at least 50% pure, at least 80% pure, or at least 90% pure.
[0200] Another form of the disclosed method generally includes:
[0201] Bringing into contact one or more analyte samples and one or
more reporter binding molecules. Each reporter binding molecule can
comprise a specific binding molecule and an amplification target
circle, and each specific binding molecule can interact with an
analyte directly or indirectly.
[0202] Separating the specific binding molecules that interact with
the analytes from the specific binding molecules that do not
interact with the analytes.
[0203] Decoupling the amplification target circles from the
reporter binding molecules that interact with the analytes.
[0204] Replicating the amplification target circles. Replication of
the amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
[0205] Another form of the disclosed method generally includes:
[0206] Bringing into contact one or more analyte samples and one or
more analyte capture agents. Each analyte capture agents can
interact with an analyte directly or indirectly.
[0207] Bringing into contact at least one of the analyte samples
and one or more reporter binding molecules. Each reporter binding
molecule can comprise a specific binding molecule and an
amplification target circle, and each specific binding molecule can
interact with an analyte capture agent directly or indirectly.
[0208] Separating the specific binding molecules that interact with
the analyte capture agents from the specific binding molecules that
do not interact with the analyte capture agents.
[0209] Decoupling the amplification target circles from the
reporter binding molecules that interact with the analyte capture
agents.
[0210] Replicating the amplification target circles. Replication of
the amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
[0211] Another form of the disclosed method generally includes:
[0212] Treating one or more analyte samples so that one or more
analytes are modified.
[0213] Bringing into contact at least one analyte samples and one
or more reporter binding molecules. Each reporter binding molecule
can comprise a specific binding molecule and an amplification
target circle, and each specific binding molecule can interact with
a modified analyte directly or indirectly.
[0214] Separating the specific binding molecules that interact with
the modified analytes from the specific binding molecules that do
not interact with the modified analytes.
[0215] Decoupling the amplification target circles from the
reporter binding molecules that interact with the modified
analytes.
[0216] Replicating the amplification target circles. Replication of
the amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding modified analytes.
[0217] Another form of the disclosed method generally includes:
[0218] Bringing into contact one or more analyte samples and a set
of analyte capture agents, a set of accessory molecules, or both,
wherein each analyte capture agent can interact with an analyte
directly or indirectly,
[0219] Bringing into contact at least one of the analyte samples
and one or more reporter binding molecules. Each reporter binding
molecule can comprise a specific binding molecule and an
amplification target circle, each specific binding molecule can
interact with an analyte directly or indirectly, and each accessory
molecule can affect the interaction of at least one of the analytes
and at least one of the specific binding molecules or at least one
of the analyte capture agents.
[0220] Separating the specific binding molecules that interact with
the analytes from the specific binding molecules that do not
interact with the analytes.
[0221] Decoupling the amplification target circles from the
reporter binding molecules that interact with the analytes.
[0222] Replicating the amplification target circles. Replication of
the amplification target circles results in the formation of tandem
sequence DNA, wherein detection of tandem sequence DNA indicates
the presence of the corresponding analytes.
[0223] The amplification target circles serve as substrates for a
rolling circle replication. This reaction requires the addition of
two reagents: (a) a rolling circle replication primer, which is
complementary to the primer complement portion of the ATC, and (b)
a rolling circle DNA polymerase. The DNA polymerase catalyzes
primer extension and strand displacement in a processive rolling
circle polymerization reaction that proceeds as long as desired,
generating a molecule of up to 100,000 nucleotides or larger that
contains up to approximately 1000 tandem copies of a sequence
complementary to the amplification target circle. A preferred
rolling circle DNA polymerase is Bst DNA polymerase.
[0224] Many different forms of RCA can be used in the disclosed
method, most of which are described in U.S. Pat. No. 5,854,033 and
WO 97/19193. For example, linear rolling circle amplification
(LRCA) involves the basic rolling circle replication of an
amplification target circle to form a strand of TS-DNA. Exponential
rolling circle amplification (ERCA) involves replication of TS-DNA
by strand displacement replication initiated at the numerous
repeated sequences in the TS-DNA. Multiple priming on both strands
of TS-DNA leads to an exponential amplification of sequences in the
amplification target circle. ERCA is preferred for the disclosed
method. If desired, the TS-DNA can be collapsed into a compact
structure for detection as described in WO 97/19193.
[0225] During rolling circle replication one may additionally
include radioactive or modified nucleotides such as
bromodeoxyuridine triphosphate, in order to label the DNA generated
in the reaction. Alternatively, one may include suitable precursors
that provide a binding moiety such as biotinylated nucleotides
(Langer et al. (1981)).
[0226] Examples of proteins that can be analyzed and detected using
the disclosed method include IL-1alpha, IL-1beta, IL-1RA, IL-2,
IL-3, IL-4, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, GROalpha,
MIP-1alpha, MIP-1beta, MCP, RANTES, MIF, G-CSF, GM-CSF, M-CSF, EGF,
FGF acidic, FGF basic, IGF-1, IGF-2, IFN-gamma, TGF-beta,
TNF-alpha, TNF-beta, TNF-RI, TNF-RII, ICAM-1, ICAM-2, IL-2Ra,
IL-4R, IL-5, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18,
IP-10, FGF-4, FGF-6, MCP-2, and MCP3.
[0227] A. Detection of Amplification Products
[0228] Amplification products can be detected directly by, for
example, primary labeling or secondary labeling, as described
below.
[0229] i. Primary Labeling
[0230] Primary labeling consists of incorporating labeled moieties,
such as fluorescent nucleotides, biotinylated nucleotides,
digoxygenin-containing nucleotides, or bromodeoxyuridine, during
strand displacement replication. For example, one may incorporate
cyanine dye deoxyuridine analogs (Yu et al., Nucleic Acids Res.,
22:3226-3232 (1994)) at a frequency of 4 analogs for every 100
nucleotides. A preferred method for detecting nucleic acid
amplified in situ is to label the DNA during amplification with
BrdUrd, followed by binding of the incorporated BrdU with a
biotinylated anti-BrdU antibody (Zymed Labs, San Francisco,
Calif.), followed by binding of the biotin moieties with
Streptavidin-Peroxidase (Life Sciences, Inc.), and finally
development of fluorescence with Fluorescein-tyramide (DuPont de
Nemours & Co., Medical Products Dept.). Other methods for
detecting nucleic acid amplified in situ include labeling the DNA
during amplification with 5-methylcytosine, followed by binding of
the incorporated 5-methylcytosine with an antibody (Sano et al.,
Biochim. Biophys. Acta 951:157-165 (1988)), or labeling the DNA
during amplification with aminoallyl-deoxyuridine, followed by
binding of the incorporated aminoallyl-deoxyuridine with an Oregon
Green.RTM. dye (Molecular Probes, Eugene, Oreg.) (Henegariu et al.,
Nature Biotechnology 18:345-348 (2000)).
[0231] Another method of labeling amplified nucleic acids is to
incorporate 5-(3-aminoallyl)-dUTP (AAdUTP) in the nucleic acid
during amplification followed by chemical labeling at the
incorporated nucleotides. Incorporated
5-(3-aminoallyl)-deoxyuridine (AAdU) can be coupled to labels that
have reactive groups that are capable of reacting with amine
groups. AAdUTP can be prepared according to Langer et al. (1981).
Proc. Natl. Acad. Sci. USA. 78: 6633-37. Other modified nucleotides
can be used in analogous ways. That is, other modified nucleotides
with minimal modification can be incorporated during replication
and labeled after incorporation.
[0232] Examples of labels suitable for addition to AAdUTP are
radioactive isotopes, fluorescent molecules, phosphorescent
molecules, enzymes, antibodies, and ligands. Examples of suitable
fluorescent labels include fluorescein isothiocyanate (FITC),
5,6-carboxymethyl fluorescein, Texas red,
nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,
rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin,
BODIPY.RTM., Cascade Blue.RTM., Oregon Green.RTM., pyrene,
lissamine, xanthenes, acridines, oxazines, phycoerythrin,
macrocyclic chelates of lanthanide ions such as quantum dye.TM.,
fluorescent energy transfer dyes, such as thiazole orange-ethidium
heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
Examples of other specific fluorescent labels include
3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine
(5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red,
Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon
Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon
Yellow 7 GLL, Atabrine, Auramine, Aurophosphine, Aurophosphine G,
BAO 9 (Bisaminophenyloxadiazole), BCECF, Berberine Sulphate,
Bisbenzamide, Blancophor FFG Solution, Blancophor SV, Bodipy F1,
Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor
RW Solution, Calcofluor White, Calcophor White ABT Solution,
Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,
Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin,
CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic
Acid), Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH--CH.sub.3,
Diamino Phenyl Oxydiazole (DAO), Dimethylamino-5-Sulphonic acid,
Dipyrrometheneboron Difluoride, Diphenyl Brilliant Flavine 7GFF,
Dopamine, Erythrosin ITC, Euchrysin, FIF (Formaldehyde Induced
Fluorescence), Flazo Orange, Fluo 3, Fluorescamine, Fura-2,
Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl
Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid, Granular Blue,
Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, Leucophor PAF,
Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),
Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue,
Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF,
MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine,
Nitrobenzoxadidole, Noradrenaline, Nuclear Fast Red, Nuclear
Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole, Pacific Blue,
Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL,
Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine,
Phycoerythrin R, Polyazaindacene Pontochrome Blue Black, Porphyrin,
Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant
Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD,
Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,
Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron
Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B,
Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene
Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can
C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R,
Thioflavin S, Thioflavin TCN, Thioflavin 5, Thiolyte, Thiozol
Orange, Tinopol CBS, True Blue, Ultralite, Uranine B, Uvitex SFC,
Xylene Orange, and XRITC.
[0233] Preferred fluorescent labels are fluorescein
(5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine
(5,6-tetramethyl rhodamine), and the cyanine dyes Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7. The absorption and emission maxima, respectively,
for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm),
Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703
mn) and Cy7 (755 run; 778 nm), thus allowing their simultaneous
detection. Other examples of fluorescein dyes include
6-carboxyfluorescein (6-FAM), 2',4', 1,4,-tetrachlorofluorescein
(TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE),
2'-chloro-5'-fluoro-7',8'-fused phenyl-1,4
-dichloro-6-carboxyfluorescein (NED), and
2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
Fluorescent labels can be obtained from a variety of commercial
sources, including Amersham Pharmacia Biotech, Piscataway, N.J.;
Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland,
Ohio.
[0234] Another useful label, related to molecular beacon
technology, is Amplifluors.TM.. Amplifluors.TM. are fluorescent
moieties and quenchers incorporated into primers containing stem
structures (usually in hairpin or stem and loop structures) such
that the quencher moiety is in proximity with the fluorescent
moiety. That is, the quencher and fluorescent are incorporated into
opposite strands of the stem structure. In the structured state,
the quencher prevents or limits fluorescence of the fluorescent
moiety. When the stem of the primer is disrupted, the quencher and
fluorescent moiety are no longer in proximity and the fluorescent
moiety produces a fluorescent signal. In the disclosed method, use
of Amplifluor.TM. primers in ERCA produces double stranded tandem
sequence DNA where the primer stem is disrupted in favor of a
complementary, replicated strand. From a reaction initially
containing structured (that is, non-fluorescent) Amplifluor.TM.
primers, fluorescence signal increases as amplification takes
place, as more and more of the Amplifluor.TM. primers are
incorporated into double stranded TS-DNA, as the Amplifluor.TM.
stems are disrupted, and as the fluorescent moieties as
consequently unquenched. Thus, use of Amplifluors.TM. is
particularly suited for real-time detection of amplification in
ERCA.
[0235] The amplification products of RCA can be detected using any
suitable technique. Real time detection, that is, detection during
the RCA reaction is a preferred mode of detection with the
disclosed method. Real time detection can be facilitated by use of
Amplifluor.TM. primers. Amplifluor.TM. primers produce a
fluorescent signal when they become incorporated into a replicated
strand and are based paired with a complementary strand.
[0236] 2. Secondary Labeling with Detection Probes
[0237] Secondary labeling consists of using suitable molecular
probes, referred to as detection probes, to detect the amplified
DNA or RNA. For example, an amplification target circle may be
designed to contain several repeats of a known arbitrary sequence,
referred to as detection tags. A secondary hybridization step can
be used to bind detection probes to these detection tags. The
detection probes may be labeled as described above with, for
example, an enzyme, fluorescent moieties, or radioactive isotopes.
By using three detection tags per amplification target circle, and
four fluorescent moieties per each detection probe, one may obtain
up to twelve fluorescent signals for every amplification target
circle repeat in the TS-DNA, yielding up to 12,000 fluorescent
moieties for every amplification target circle that is amplified by
RCA.
[0238] 3. Multiplexing and Hybridization Array Detection
[0239] RCA is easily multiplexed by using sets of different
amplification target circles, each set carrying different address
tag sequences designed for binding to unique address probes. Note
that although the address tag sequences for each amplification
target circle are different, the primer complement portion may
remain unchanged, and thus the primer for rolling circle
replication can remain the same for all targets. The TS-DNA
molecules generated by RCA are of high molecular weight and low
complexity; the complexity being the length of the amplification
target circle. A given TS-DNA can be captured to a fixed position
in a solid support by, for example, including within the spacer
region of the amplification target circles a unique address tag
sequence for each unique amplification target circle. TS-DNA
generated from a given amplification target circle will then
contain sequences corresponding to a specific address tag
sequence.
[0240] 4. Combinatorial Multicolor Coding
[0241] A preferred form of multiplex detection involves the use of
a combination of labels that either fluoresce at different
wavelengths or are colored differently. One of the advantages of
fluorescence for the detection of hybridization probes is that
several targets can be visualized simultaneously in the same
sample. Using a combinatorial strategy, many more targets can be
discriminated than the number of spectrally resolvable
fluorophores. Combinatorial labeling provides the simplest way to
label probes in a multiplex fashion since a probe fluor is either
completely absent (-) or present in unit amounts (+); image
analysis is thus more amenable to automation, and a number of
experimental artifacts, such as differential photobleaching of the
fluors and the effects of changing excitation source power
spectrum, are avoided.
[0242] The combinations of labels establish a code for identifying
different detection probes and, by extension, different analytes to
which those detection probes are associated with. This labeling
scheme is referred to as Combinatorial Multicolor Coding (CMC).
Such coding is described by Speicher et al., Nature Genetics
12:368-375 (1996). Any number of labels, which when combined can be
separately detected, can be used for combinatorial multicolor
coding. It is preferred that 2, 3, 4, 5, or 6 labels be used in
combination. It is most preferred that 6 labels be used. The number
of labels used establishes the number of unique label combinations
that can be formed according to the formula 2.sup.N-1, where N is
the number of labels. According to this formula, 2 labels forms
three label combinations, 3 labels forms seven label combinations,
4 labels forms 15 label combinations, 5 labels form 31 label
combinations, and 6 labels forms 63 label combinations.
[0243] Speicher et al. describes a set of fluors and corresponding
optical filters spaced across the spectral interval 350-770 nm that
give a high degree of discrimination between all possible fluor
pairs. This fluor set, which is preferred for combinatorial
multicolor coding, consists of 4'-6-diamidino-2-phenylinodole
(DAPI), fluorescein (FITC), and the cyanine dyes Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7. Any subset of this preferred set can also be used
where fewer combinations are required. The absorption and emission
maxima, respectively, for these fluors are: DAPI (350 nm; 456 nm),
FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588
nm), Cy5 (652 nm; 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm;
778 nm). The excitation and emission spectra, extinction
coefficients and quantum yield of these fluors are described by
Ernst et al., Cytometry 10:3-10 (1989), Mujumdar et al., Cytometry
10:11-19 (1989), Yu, Nucleic Acids Res. 22:3226-3232 (1994), and
Waggoner, Meth. Enzymology 246:362-373 (1995). These fluors can all
be excited with a 75W Xenon arc.
[0244] B. Further Amplification
[0245] Secondary DNA strand displacement is another way to amplify
TS-DNA. Secondary DNA strand displacement is accomplished by
hybridizing secondary DNA strand displacement primers to TS-DNA and
allowing a DNA polymerase to synthesize DNA from these primed
sites. The product of secondary DNA strand displacement is referred
to as secondary tandem sequence DNA or TS-DNA-2. Secondary DNA
strand displacement and strand displacement cascade amplification
are described in U.S. Pat. No. 5,854,033 and WO 97/19193. Strand
displacement cascade amplification, also referred to as exponential
rolling circle amplification (ERCA) is a preferred form of RCA for
use in the disclosed method.
[0246] In exponential RCA, a secondary DNA strand displacement
primer primes replication of TS-DNA to form a complementary strand
referred to as secondary tandem sequence DNA or TS-DNA-2. As a
secondary DNA strand displacement primer is elongated, the DNA
polymerase will run into the 5' end of the next hybridized
secondary DNA strand displacement molecule and will displace its 5'
end. In this fashion a tandem queue of elongating DNA polymerases
is formed on the TS-DNA template. As long as the rolling circle
reaction continues, new secondary DNA strand displacement primers
and new DNA polymerases are added to TS-DNA at the growing end of
the rolling circle. A tertiary DNA strand displacement primer
strand (which is complementary to the TS-DNA-2 strand and which can
be the rolling circle replication primer) can then hybridize to,
and prime replication of, TS-DNA-2 to form TS-DNA-3 (which is
equivalent to the original TS-DNA). Strand displacement of TS-DNA-3
by the adjacent, growing TS-DNA-3 strands makes TS-DNA-3 available
for hybridization with secondary DNA strand displacement primer.
This results in another round of replication resulting in TS-DNA-4
(which is equivalent to TS-DNA-2). TS-DNA-4, in turn, becomes a
template for DNA replication primed by tertiary DNA strand
displacement primer. The cascade continues this manner until the
reaction stops or reagents become limiting. The additional forms of
tandem sequence DNA beyond secondary tandem sequence DNA are
collectively referred to herein as higher order tandem sequence
DNA. Higher order tandem sequence DNA encompasses TS-DNA-3,
TS-DNA-4, and any other tandem sequence DNA produced from
replication of secondary tandem sequence DNA or the products of
such replication. In a preferred mode of ERCA, the rolling circle
replication primer serves as the tertiary DNA strand displacement
primer, thus eliminating the need for a separate primer.
Exponential rolling circle amplification is further described in
U.S. Pat. No. 5,854,033 and WO 97/19193 (where it is referred to as
strand displacement cascade amplification).
[0247] Illustrations
[0248] The disclosed method can be further described by the
following illustrations.
[0249] One form of the disclosed method involving the use of circle
capture probes for the detection of HIV p24 antigen is described
below.
[0250] Microtiter plates will be pre-coated with mouse anti-HIV p24
antibody. Incubation of sample with HIV p24 with antibody-coated
microtiter plates will result in the binding of HIV p24 antigen to
antibodies anchored on to the plates. Plate bound HIV p24 antigen
will then be recognized by polyclonal anti-HIV p24 goat antibody
that has been conjugated with the amplification target circle (the
conjugate is the reporter binding molecule) and has been
preannealed to a RCA circle. Subsequent to washing, captured
amplification target circles will be released (decoupled) during
ERCA amplification using appropriate primers. RCA signals will be
detected with either a plate reader or ABI-7700 real time
instrument and using Amplifluors.TM. or molecular beacons.
[0251] Another form of the disclosed method involving the use of
circle linkers for the detection of antigens is described
below.
[0252] Microtiter plates will be pre-coated with appropriate
capture antibodies, in an arrayed fashion, for analyte detection.
Incubation of test samples will result in the binding of specific
analyte to antibodies anchored on to the plates. Plate bound
analytes will then be recognized by a detector antibody that has
been conjugated with the amplification target circle (the conjugate
is the reporter binding molecule) via a cleavable linker (that is,
a circle linker). Subsequent to washing, antibody-conjugated
amplification target circle will be released (decoupled), inside a
microtiter plate, by cleaving the linker (for example, by DTT
treatment to cleave disulfide linkage). Released amplification
target circle will be used for signal amplification by ERCA. The
signal detection will be carried out with either a real time assay
instrument (ABI 7700 sequence detector) or a plate reader using
Amplifluors.TM. or molecular beacons.
EXAMPLES
[0253] The disclosed method can be further described, and relevant
principles illustrated, through the following examples.
A. Example 1
Coating of Micro Amp Polypropylene Tubes with Antibody
[0254] This example demonstrates that Micro Amp tubes can be coated
with antibody as efficiently as microtiter plates. Micro Amp
polypropylene tubes (appropriate for use in ABI 7700 sequence
detector) and polystyrene ELISA microtiter plates were coated with
variable amounts of anti-biotin antibody. For this purpose, 40
.mu.l of desired antibody, in 50 mM carbonate buffer pH 9.6, was
incubated overnight at 4.degree. C. in these tubes. Subsequent to
incubation, any uncoated material was washed with 150 mM phosphate
buffer saline, pH 7.2. Subsequent to washing, coated anti-biotin
antibody was recognized by 1 .mu.g/ml of biotin coupled horse
reddish peroxidase (HRP). Subsequent to washing of unbound
proteins, the relative amounts of bound HRP were detected by using
OPD assay. After 10-15 min, the assay mixture was transferred from
Micro Amp tubes to Costar plates and the absorbance of the assay
mixture was evaluated at 450 nm using a plate reader. As shown in
FIG. 2, both microtiter plates and Micro Amp tubes showed similar
levels of antibody coating.
B. Example 2
Detection of Amplification Target Circles, Captured on
Antibody-Coated Micro Amp Tubes, by Immuno ERCA Using ABI 7700
Sequence Detector Instrument
[0255] This example demonstrates amplification and detection of
captured amplification target circles. The strategy for this
example is shown in FIG. 3. In this example, variable amounts of
reporter binding molecules were used to assess, in part, the effect
of the amount of reporter binding molecules on signal over
background. Micro Amp tubes were coated, as described in Example 1,
with 30 .mu.l of 10 .mu.g/ml of either anti-biotin antibody or
mouse IgG. These antibodies serve as "analytes" in this example.
Subsequent to washing, the tubes were blocked using blocking
solution (blocker casein in PBS, Pierce Chemicals) and washed again
with PBS carrying 0.05% Tween 20. In a separate tube, amplification
target circle 1822oc88 was annealed to 3'-biotin labeled circle
capture probe in 2.times.SSC. The amplification target
circle/circle capture probe/biotin is a reporter binding molecule.
The biotin is the specific binding molecule. Various amounts of
circle capture probe annealed circles (freshly diluted in 30 .mu.l
PBS) were added to coated Micro Amp tubes and incubated at
37.degree. C. for 1 hour. Unbound probe-annealed circles as well as
probes were washed away using PBS. Trapped circles were detected by
ERCA using TET linked Amplifluor.TM. as one of the two primers and
real time detection of fluorescence in ABI 7700 sequence detector.
This allowed real-time detection of amplification products as TET
moieties emitted fluorescent signal after synthesis of
complementary strands. 30 .mu.l of the ERCA mix contained 5%
tetramethyl ammonium oxlate, 400 .mu.M dNTP mix, 1 .mu.M each of
the two primers, 8 units of Bst DNA polymerase (New England
Biolabs, Mass.), and 1.times. modified ThermoPol buffer containing
20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH.sub.4).sub.2SO4 and
0.1% Triton X-100. ERCA reactions were performed at 60.degree. C.
Histographic analysis of the amplification results is shown in FIG.
4. The graphs show fluorescence detected over time (the time units
are labeled as "cycles" in the graphs although there was no cycling
involved). Fluorescent signal is observed in assays containing the
anti-biotin antibody "analyte" prior to signal seen in the control
assays without analyte. FIG. 4 notes this difference in signal
appearance as .DELTA.Ct. As can be seen, even when very few
reporter binding molecules (360) were used, there was still an
easily observable difference in the time of signal appearance.
C. Example 3
Detection of the Binding of Variable Number of Amplification Target
Circles Using Fixed Number of Amplification Target Circles in
Immuno ERCA with Amplifluors.TM.
[0256] This example demonstrates detection binding of a fixed
number of amplification target circles to a variable number of
circle capture probes. The strategy for this example is shown in
FIG. 5. Because the number of amplification target circles used
were the same, the background caused by the circles was expected to
be similar in all of the assays. Anti biotin antibody-coated or
mouse IgG-coated Micro Amp tubes were used to incubate various
amounts of 3'-biotin labeled circle capture probes, in PBS.
Subsequent to the removal of unbound probes, 1.times.10.sup.6
1822oc88 amplification target circles, in 2.times. SSC, were used
for annealing to antibody-bound circle capture probes at 37.degree.
C. for 1 hour. Subsequent to the washing of excess circles,
probe-annealed circles were detected by ERCA as indicated before.
Amplification products were detected in real time as TET moieties
emitted fluorescent signal after synthesis of complementary
strands. Difference in Ct values between anti biotin
antibody-coated and mouse IgG-coated tubes at various for various
amounts of circle capture probes are plotted in FIG. 6. The graph
shows the difference in the time of fluorescent signal detection
(.DELTA.Ct) using different amounts of circle capture probes. As
can be seen, there was an easily observable difference in the time
of signal appearance at all amounts of circle capture probe. These
results also demonstrate that variations in the circle capture
probe, bound to capture antibodies, can be successfully detected by
immuno-ERCA.
D. Example 4
Detection of IL-8 Using Immuno-ERCA (ERCA-ELISA)
[0257] This example demonstrates use of a form of the disclosed
method to detect IL-8. The strategy for this example is shown in
FIG. 7. Micro Amp tubes were coated with 40 .mu.l of 10 .mu.g/ml
anti-IL-8 mouse mAb in 50 mM carbonate buffer, pH 9.6, at 4.degree.
C. for 12 hrs. Variable concentrations of IL-8 (40 .mu.l) were
incubated in these tubes, at 37.degree. C., for 1 hr. Subsequent to
the washing of unbound IL-8 molecules, 1 .mu.g/ml of biotinylated
anti-IL-8 secondary antibody (40 .mu.l) was incubated at 37.degree.
C. for 1 hr. Subsequent to washing, the tubes were incubated with
40 .mu.l of 10 ng/ml anti-biotin antibody that has been covalently
conjugated with circle capture probe via its 3' end and is
pre-annealed with the 1822in88 amplification target circle at
37.degree. C. for 5 hrs. Trapped circles were detected by ERCA
using FAM Amplifluors.TM. as described before. This allowed
real-time detection of amplification products as FAM moieties
emitted fluorescent signal after synthesis of complementary
strands. Differences in .DELTA.Ct values between no IL-8 and
various concentrations of IL-8, used in this assay, are plotted in
FIG. 8. The graph shows the difference in the time of fluorescent
signal detection (.DELTA.Ct) when different amounts of IL-8 were
present. As can be seen, the .DELTA.Ct increases steadily as the
amount of IL-8 increased.
[0258] It is understood that the disclosed invention is not limited
to the particular methodology, protocols, and reagents described as
these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims.
[0259] It must be noted that as used herein and in the appended
claims, the singular forms "a ", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to "the antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0260] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are as
described. Publications cited herein and the material for which
they are cited are specifically incorporated by reference. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0261] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *